Detection method of genotoxic impurity methyl bromoacetate

Abstract

The invention provides a method for detecting genotoxic impurity methyl bromoacetate in a compound 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1, 2, 4-triazole-3-yl)-sulfur) acetic acid, which comprises the following steps: reacting the compound, a solvent, triethylamine and a derivatization reagent to obtain a test solution; the compound is-((5-bromo-4-(2-chloro-4-cyclopropyl naphthyl-1-yl)-4H-1, 2, 4-triazole-3-yl)-sulfur) acetic acid, and the structural formula of the compound is shown in the description. Methyl bromoacetate, a solvent, triethylamine and a derivatization reagent are subjected to a reaction, and a reference substance solution is obtained; and respectively performing high performance liquid chromatography detection on the test solution and the reference solution to obtain a test chromatogram and a reference chromatogram. The method provided by the invention is good in specificity and good in durability. The method provided by the invention can be used for accurately detecting each genotoxic impurity in the raw material medicine or the preparation, the separation degree of each peak, the number of theoretical plates and the symmetry factor are good, and the response is high.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on August 29, 2023, with application number 202311106930.0 and invention name “Method for detecting the genotoxic impurity methyl bromoacetate”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention relates to the field of detection technology, and in particular to a method for detecting methyl bromoacetate, a genotoxic impurity. Background Art

[0003] Compound 1 is a targeted URAT1 inhibitor with a chemical name of 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid. The prior arts WO2017215589A1, WO2019114838A1 and WO2021249468A1 respectively disclose the structure, crystal form and preparation method of compound 1.

[0004]

[0005] In December 2015, the US FDA approved AstraZeneca's first targeted URAT1 inhibitor, Lesinurad, and approved its 200 mg / day dose in combination with xanthine oxidase inhibitors XOI (such as Febuxostat, etc.) for the treatment of hyperuricemia and gouty arthritis. However, for long-term medication for a metabolic disease, drug safety issues are particularly important.

[0006] Therefore, it is very necessary to accurately reflect the content of the genotoxic impurity methyl bromoacetate in the raw material or preparation of Compound 1 and develop a detection method for the genotoxic impurity methyl bromoacetate. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a detection method for the genotoxic impurity methyl bromoacetate. The detection method provided by the present invention has good separation, specificity and durability, high responsiveness and accurate results.

[0008] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0009] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0010] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0011] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the following processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0012] In order to accurately reflect the content of genotoxic impurities in the raw materials or preparations of compound 1, a reasonable basis can be provided for the formulation of quality standards, so as to better control and master product quality, thereby improving the safety of clinical medication, the present invention provides a method for detecting compound 1 and genotoxic impurity methyl bromoacetate. The detection method has high accuracy and precision, strong durability, and is suitable for the inspection and quality control of genotoxic impurities of compound 1 in the pharmaceutical industry.

[0013] The present invention provides a method for detecting methyl bromoacetate, a genotoxic impurity in the compound 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid, characterized in that the method comprises the following steps:

[0014] A) reacting a compound, a solvent, triethylamine and a derivatizing agent to obtain a test solution; the compound is -((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid;

[0015] B) reacting methyl bromoacetate, a solvent, triethylamine and a derivatization agent to obtain a reference substance solution;

[0016] C) subjecting the test solution and the reference solution to high performance liquid chromatography, respectively, to obtain a test sample chromatogram and a reference sample chromatogram;

[0017] The specific chromatographic parameters are as follows:

[0018] Mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile;

[0019] The elution procedure is:

[0020] 0-7min, phase A 54%, phase B 46%;

[0021] 7-11min: phase A 54-52%, phase B 46%-48%;

[0022] 11-18min: Phase A 52-50%, Phase B 48-50%;

[0023] 18-20min: 50% phase A, 50% phase B;

[0024] 20-22min: Phase A 50%-5%, Phase B 50%-95%;

[0025] 22-28min: Phase A 5%, Phase B 95%;

[0026] 28-28.1min: Phase A 5%-54%, Phase B 95%-46%;

[0027] 28.1-35min: phase A 54%, phase B 46%;

[0028] D) calculating the content of methyl bromoacetate by external standard method according to the concentration of the reference substance, the peak area of ​​the reference substance solution in the chromatogram, and the peak area of ​​the component corresponding to the reference substance solution in the test solution, and based on the detection conditions in step C).

[0029] The present invention provides a method for detecting the genotoxic impurity methyl bromoacetate in the compound 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid, which comprises first reacting the compound, a solvent, triethylamine and a derivatization reagent to obtain a test solution.

[0030] According to the present invention, after the compound is dissolved in a solvent, triethylamine and a derivatization reagent are added, mixed, reacted, and allowed to stand to obtain a test solution.

[0031] The compound of the present invention is -((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid.

[0032] The structure is shown in formula (I):

[0033]

[0034] In some preferred embodiments of the present invention, the derivatization agent is tetraphenylthiophenol.

[0035] The inventors creatively discovered that when tetraphenylthiophenol is used as a derivatization reagent, the impurities in the derivatization reagent have minimal interference with the target derivatization peak.

[0036] The solvent of the present invention includes but is not limited to acetonitrile.

[0037] In some preferred embodiments of the present invention, the reaction temperature is 25-35° C. and the reaction time is 6-55 min.

[0038] In some preferred embodiments of the present invention, the reaction temperature is 25-35° C. and the reaction time is 8-40 min.

[0039] In some preferred embodiments of the present invention, the reaction temperature is 25-33° C. and the reaction time is 8-13 min.

[0040] In some preferred embodiments of the present invention, the reaction temperature is 25-30° C. and the reaction time is 8-10 min.

[0041] The present inventors creatively discovered that within the above derivatization temperature and time, the derivatization result is good.

[0042] In some preferred embodiments of the present invention, the mass-volume ratio of the compound, solvent, triethylamine and derivatization agent is (10-100 mg): 7 mL: (40-60 μL): (25-40 μL).

[0043] In some preferred embodiments of the present invention, the mass-volume ratio of the compound, solvent, triethylamine and derivatization agent is (10-100 mg): 7 mL: (45-60 μL): (25-35 μL).

[0044] In some preferred embodiments of the present invention, the mass-volume ratio of the compound, solvent, triethylamine and derivatization agent is (10-100 mg): 7 mL: 50 μL: 25 μL.

[0045] Within the above-mentioned ratio range, the derivatization of the present invention is complete, there is no significant difference in peak area, and the target peak separation is good.

[0046] Methyl bromoacetate, a solvent, triethylamine and a derivatization reagent are reacted to obtain a reference solution.

[0047] The solvent of the present invention includes but is not limited to acetonitrile.

[0048] According to the present invention, the above reaction is preferably carried out at 25-35°C for 6-55 minutes; preferably for 8-15 minutes; more preferably for 8-13 minutes at 25-33°C; most preferably for 8 minutes at 25°C-30°C.

[0049] The experimental results show that the concentration of methyl bromoacetate of the present invention is in a good linear relationship between 21.39 ng / ml and 213.88 ng / ml.

[0050] The present invention also includes the preparation of the sensitive solution, specifically: after the compound and methyl bromoacetate are dissolved in a solvent, triethylamine is added, and the derivatization reagent and the reference substance are mixed and reacted to obtain the sensitive solution. The solvent includes but is not limited to acetonitrile.

[0051] The test solution and the reference solution are respectively subjected to high performance liquid chromatography to obtain a test sample chromatogram and a reference sample chromatogram; according to the concentration of the reference sample, the peak area of ​​the reference sample solution in the chromatogram, and the peak area of ​​the component corresponding to the reference sample solution in the chromatogram, and based on the detection conditions in step C), the content of methyl bromoacetate is calculated by an external standard method.

[0052] The present invention does not limit the above-mentioned external standard method for calculating the content of methyl bromoacetate, and it can be well known to those skilled in the art.

[0053] The specific chromatographic parameters of the present invention are as follows:

[0054] Mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile;

[0055] The elution procedure is:

[0056] 0-7min, phase A 54%, phase B 46%;

[0057] 7-11min: phase A 54-52%, phase B 46%-48%;

[0058] 11-18min: Phase A 52-50%, Phase B 48-50%;

[0059] 18-20min: 50% phase A, 50% phase B;

[0060] 20-22min: Phase A 50%-5%, Phase B 50%-95%;

[0061] 22-28min: Phase A 5%, Phase B 95%;

[0062] 28-28.1min: Phase A 5%-54%, Phase B 95%-46%;

[0063] 28.1-35min: phase A 54%, phase B 46%;

[0064] The present invention achieves good baseline separation, good separation of each peak and stable baseline by selecting the mobile phase gradient.

[0065] In the present invention, the chromatographic column is ACE EXCEL 3C18-PFP, the chromatographic column specifications are 4.6×150 mm, 3 μm; and the chromatographic column temperature is 45°C.

[0066] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above 45°C condition.

[0067] In the present invention, the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 to 100 μL; preferably 20 μL.

[0068] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 1 ml / min, which was the most preferred solution.

[0069] In the present invention, the detection wavelength is 278 nm.

[0070] In the present invention, the detection limit concentration of methyl bromoacetate is 10.69 ng / ml, that is, 0.2138 ng); the quantification limit concentration is 21.39 ng / ml, that is, 0.4278 ng).

[0071] The invention has low detection limit, low quantitative limit, high separation degree and good effect.

[0072] The invention provides a method for detecting methyl bromoacetate, a genotoxic impurity in a compound 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid, comprising the following steps: A) reacting a compound, a solvent, triethylamine and a derivatization reagent to obtain a test solution; the compound is methyl bromoacetate, a genotoxic impurity in a compound 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid, and the method comprises the following steps: A) reacting a compound, a solvent, triethylamine and a derivatization reagent to obtain a test solution; the compound is methyl bromoacetate, a genotoxic impurity in a compound 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid. ) acetic acid; B) reacting methyl bromoacetate, solvent, triethylamine and a derivatizing agent to obtain a reference solution; C) subjecting the test solution and the reference solution to high performance liquid chromatography, respectively, to obtain a test chromatogram and a reference chromatogram; D) calculating the content of methyl bromoacetate by an external standard method according to the concentration of the reference substance, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the component corresponding to the reference solution in the test solution, and based on the detection conditions in step C). The method provided by the present invention has good specificity and good durability. The method provided by the present invention can accurately detect various genotoxic impurities in bulk drugs or preparations, and the separation degree, theoretical plate number and symmetry factor of each peak are good, and the response is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 It is a specific overlay map;

[0074] Figure 2 is a linear relationship graph;

[0075] Figure 3 It is the chromatogram of Comparative Example 4;

[0076] Figure 4 This is the chromatogram of Comparative Example 5. DETAILED DESCRIPTION

[0077] The present invention provides a method for detecting a genotoxic impurity methyl bromoacetate, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0078] To further illustrate the present invention, a method for detecting a genotoxic impurity methyl bromoacetate provided by the present invention is described in detail below in conjunction with examples.

[0079] The batch number of the methyl bromoacetate of the present invention is 20180410, which is from WuXi AppTec New Drug Development Co., Ltd.; the batch number of triethylamine is K1806-2 Aladdin

[0080] Compound 1, batch number H21715-31-P1, was obtained from WuXi AppTec.

[0081] Example 1 Detection Method of Genotoxic Methyl Bromoacetate Impurity

[0082] The preparation process of compound 1 (see WO2021249468A1 for details) uses methyl bromoacetate, which has a risk of genotoxicity. In order to ensure the safety of the finished product, a sensitive analytical method was developed to detect its residues.

[0083] The ultraviolet absorption of methyl bromoacetate is weak, and it is difficult to directly measure it using HPLC or GC methods. Therefore, a derivatization method is used to measure the methyl bromoacetate derivative with strong ultraviolet absorption, thereby determining the content of methyl bromoacetate.

[0084] By examining different types and amounts of derivatization reagents, the amount of triethylamine, the reaction temperature and reaction time, optimizing the elution conditions, column temperature, etc., and examining the separation degree between the derivatization reagent and adjacent impurities, the analytical method for the detection of methyl bromoacetate was finally determined. The chromatographic conditions are shown in Table 1.

[0085] Table 1. Chromatographic conditions

[0086]

[0087] (I) Screening of derivatization reaction conditions

[0088] (1) Preparation of stock solution

[0089] 4-Phenylthiophenol stock solution: Take 25 mg of 4-phenylthiophenol into a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, mix well, and prepare a concentration of 1 mg / ml.

[0090] Triethylamine solution: Measure 1 ml of triethylamine into a 10 ml volumetric flask, add acetonitrile to dilute to the scale, and shake well.

[0091] Formic acid solution: Measure 1 ml into a 10 ml volumetric flask, add acetonitrile to dilute to the mark, shake well, and you have it.

[0092] Methyl bromoacetate stock solution ①: Take 10 mg of bromoacetic acid and put it into a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use this as methyl bromoacetate stock solution ① with a concentration of 1 mg / ml.

[0093] Methyl bromoacetate stock solution ②: Measure 10 μl of bromoacetic acid stock solution ① into a 10 ml volumetric flask, add acetonitrile to dilute to the scale, and shake well to obtain methyl bromoacetate stock solution ② with a concentration of 1 μg / ml.

[0094] Test sample stock solution: Take 10 mg of the test sample into a 2 ml injection vial and add 1 ml of acetonitrile to dissolve it to a concentration of 10 mg / ml.

[0095] (2) Investigation of reaction time

[0096] Preparation of the test sample spiked derivatization solution: 725 μl of acetonitrile, 100 μl of the test sample stock solution, 100 μl of methyl bromoacetate stock solution ②, 25 μl of 4-phenylthiophenol stock solution, and 50 μl of triethylamine were added to the same 2 ml injection vial. After mixing, let stand at room temperature for 8 min and then perform continuous injection detection (the detection time is 47 min).

[0097] Different reaction times Target peak area 8min 14974 55min 14979

[0098] The test results show that the derivatization solution reacts completely after standing for 8 minutes at room temperature, so the reaction time is set at 8 minutes.

[0099] (3) Investigation of different alkali amounts

[0100] Preparation of the spiked derivatization solution of the test sample with different alkali addition amounts: Take an appropriate amount of acetonitrile, 100 μl of the test sample stock solution, 100 μl of methyl bromoacetate stock solution ②, 25 μl of 4-phenylthiophenol stock solution, and different volumes of triethylamine solution into the same 2 ml injection vial, mix well, let stand at room temperature for 10 minutes, and then inject and test. Among them, the amount of acetonitrile added is adjusted according to the amount of triethylamine solution added to keep the final derivatization solution volume consistent.

[0101] Different alkali addition amount (μl) Target peak area 40 14213 50 14974 60 14414

[0102] The experimental results showed that adjusting the amount of triethylamine solution added had no significant effect on the area of ​​the derived target peak, and the amount of base was set at 50 μl for subsequent condition optimization.

[0103] (4) Investigation of the amount of derivatization reagent used

[0104] Preparation of the spiked derivatization solution with different amounts of derivatization reagents: Take an appropriate amount of acetonitrile, 100 μl of the sample stock solution, 100 μl of methyl bromoacetate stock solution ②, different volumes of 4-phenylthiophenol stock solution, and 50 μl of triethylamine solution into the same 2 ml injection vial, mix well, let stand at room temperature for 10 min, and then inject and test. The amount of acetonitrile added is adjusted according to the amount of 4-phenylthiophenol stock solution added to keep the final derivatization solution volume consistent.

[0105] Amount of different derivatization reagents (μl) Target peak area 25 14974 40 14637

[0106] The experimental results show that increasing the amount of derivatization reagent has no obvious effect on the derivatization target peak area, and the derivatization reaction is complete. Considering that the derivatization reagent needs to be invested in excess, the amount of derivatization reagent is not further reduced and optimized, and the amount of derivatization reagent is set at 25μl.

[0107] (5) Adjustment of sampling method

[0108] Since the 10mg / ml test sample stock solution requires a large amount of test sample, the sample preparation method was adjusted, and the test sample solution was directly prepared into 1mg / ml in a volumetric flask for derivatization reaction. All solutions were simultaneously amplified to a volumetric flask for derivatization reaction according to the condition optimization results. Considering the reaction in the volumetric flask, acetonitrile was used to make the final volume, so the triethylamine-acetonitrile solution was directly converted into the volume of added triethylamine according to the amount of triethylamine. After peak area comparison, there was no significant difference in the target peak area derived from the same concentration by the two sample preparation methods, and the specificity was good. The blank derivatization solution and the test sample derivatization solution had no significant interference on the target peak. The peak area of ​​the reference sample derivatization solution was close to that of the spiked test sample derivatization solution, and the recovery rate was normal.

[0109] Example 2

[0110] 2.1 Preparation of stock solution:

[0111] 4-Phenylthiophenol stock solution-1: Take 25.03 mg of 4-phenylthiophenol, accurately weigh it into a 25 ml brown volumetric bottle, add acetonitrile to dissolve and dilute to the scale, and shake well;

[0112] 4-Phenylthiophenol stock solution-2: Take 10.51 mg of 4-phenylthiophenol, accurately weigh it into a 25 ml brown volumetric bottle, add acetonitrile to dissolve and dilute to the scale, and shake well;

[0113] Prepare stock solutions of methyl bromoacetate with concentrations of 9.7414 μg / mL to 10.1441 μg / mL respectively.

[0114] 2.2 Configuration of proprietary solution:

[0115] Blank: acetonitrile solution;

[0116] Sys-1: Accurately pipette 7 mL of acetonitrile, 50 μL of triethylamine, and 250 μL of 4-phenylthiophenol-1 into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0117] Sys-2: Accurately pipette 7 mL of acetonitrile, 100 μL of methyl bromoacetate stock solution (10.1244 μg / L), 50 μL of triethylamine, and 250 μL of 4-phenylthiophenol-1 into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0118] Sys-3: Accurately weigh 10.069 mg of compound 1 into a 10 mL brown volumetric flask, transfer 7 mL of acetonitrile to dissolve it, add 250 μL of 4-phenylthiophenol-1 and 50 μL of triethylamine to the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min to obtain Sys-3.

[0119] Sys-4: Accurately weigh 10.120 mg of compound 1 into a 10 mL brown volumetric flask, transfer 7 mL of acetonitrile to dissolve it, add 100 μL of 10.1244 μg / L methyl bromoacetate stock solution, 250 μL of 4-phenylthiophenol-1, and 50 μL of triethylamine into the same 10 mL brown volumetric flask, add acetonitrile to the volume, mix well, and react at room temperature for 10 min.

[0120] 2.3 Preparation of stability solution

[0121] Blank: acetonitrile solution;

[0122] Sta-Std: Accurately pipette 7 mL of acetonitrile, 100 μL of methyl bromoacetate stock solution (10.1244 μg / L), 50 μL of triethylamine, and 250 μL of 4-phenylthiophenol-1 into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0123] Sta-spl: accurately weigh 10.120 mg of compound 1 into a 10 mL brown volumetric flask, transfer 7 mL of acetonitrile to dissolve it, add 100 μL of 10.1244 μg / L of methyl bromoacetate stock solution, 250 μL of 4-phenylthiophenol-1, and 50 μL of triethylamine into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0124] 2.4 Configuration of precision solution:

[0125] Blank: acetonitrile solution;

[0126] Std-1: Accurately pipette 7 mL of acetonitrile, 100 μL of methyl bromoacetate stock solution 10.1244 μg / L, 50 μL of triethylamine, and 250 μL of 4-phenylthiophenol-1 into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0127] Std-2: Accurately pipette 7 mL of acetonitrile, 100 μL of methyl bromoacetate stock solution (10.1441 μg / L), 50 μL of triethylamine, and 250 μL of 4-phenylthiophenol-1 into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0128] 2.5 Accuracy and Repeatability Solution Configuration:

[0129] Std-1: Accurately pipette 7 mL of acetonitrile, 100 μL of methyl bromoacetate stock solution 10.1244 μg / L, 50 μL of triethylamine, and 250 μL of 4-phenylthiophenol-1 into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0130] Unsampled test solution: Take about 10 mg of compound 1, weigh accurately, place in a 10 ml brown volumetric flask, add 7 ml acetonitrile to dissolve, then accurately pipette 50 μL of triethylamine and 250 μL of 4-phenylthiophenol-1 into the same 10 ml brown volumetric flask, add acetonitrile to the volume, mix well, and react at room temperature for 10 min. Prepare 2 copies in parallel.

[0131] Sample addition - low concentration: Take about 10 mg of compound 1, accurately weigh it, and place it in a 10 ml brown volumetric flask, add 50 μL of 10.1244 μg / L methyl bromoacetate stock solution, 50 μL of triethylamine, and 1250 μL of 4-phenylthiophenol to the same 10 ml brown volumetric flask, add acetonitrile to the volume, mix well, and react at room temperature for 10 minutes.

[0132] Sample addition - medium concentration: take about 10 mg of compound 1, accurately weigh it, and place it in a 10 ml brown volumetric flask, add 100 μL of methyl bromoacetate stock solution 10.1244 μg / L, 50 μL of triethylamine, and 1250 μL of 4-phenylthiophenol to the same 10 ml brown volumetric flask, add acetonitrile to the volume, mix well, and react at room temperature for 10 minutes.

[0133] Sample addition - high concentration: Take about 10 mg of compound 1, weigh accurately, and place in a 10 ml brown volumetric flask, add 150 μL of 10.1244 μg / L methyl bromoacetate stock solution, 50 μL of triethylamine, and 1250 μL of 4-phenylthiophenol to the same 10 ml brown volumetric flask, add acetonitrile to the volume, mix well, and react at room temperature for 10 minutes.

[0134] 2.6 Detection line and quantitative limit solution configuration:

[0135] Detection limit solution: Accurately pipette 7 ml of acetonitrile, 10 μL of methyl bromoacetate stock solution 10.6940 μg / L, 50 μL of triethylamine, and 250 μL of 4-phenylthiophenol stock solution-2 into the same 10 ml brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0136] Quantitation limit solution: Accurately pipette 7 ml of acetonitrile, 20 μL of methyl bromoacetate stock solution 10.6940 μg / L, 50 μL of triethylamine, and 250 μL of 4-phenylthiophenol stock solution-2 into the same 10 ml brown volumetric flask, add acetonitrile to make up to volume, mix well, and react at room temperature for 10 min.

[0137] 2.7 Linear solution configuration:

[0138] Prepare methyl bromoacetate solutions with concentrations of 21.388 to 213.8796 ng / mL in the same manner as above;

[0139] 2.8 Use the chromatographic conditions in Table 1 to verify the methodology.

[0140] 2.8.1 Exclusivity

[0141] Take the specific solution, inject it separately, and record the chromatogram. Record the retention time of the derivative product and use the retention time to locate the impurity. The results are shown in Table 6-1. Among them: Sys-4 refers to Sta-spl 0 hour data. The specificity overlay diagram is shown in Figure 1 , Figure 1 This is a specificity overlay map, from top to bottom: sys1, sys2, sys3, sys4.

[0142] Conclusion: The blank solvent does not interfere with the target chromatographic peak; the blank derivatization solution has no interference at the target chromatographic peak and the minimum separation between the target chromatographic peak and nearby impurity peaks is 1.9, and the method has good specificity.

[0143] 2.8.2 Injection precision

[0144] The precision solution was injected 6 times in succession. The results of the precision solution injection are shown in Table 6.

[0145] Table 6 Injection precision results

[0146]

[0147] Conclusion: The RSD of the target peak area after 6 consecutive injections of the solution was 1.2%, and the injection precision was good.

[0148] 2.8.3 Solution stability

[0149] The stability solution stored at room temperature was sampled and the changes in the content of methyl bromoacetate in the reference solution and the spiked test solution within 17.5 hours compared with 0 hours were investigated. The system suitability results are shown in Table 7 and the stability results are shown in Table 8. Among them, Std 0h refers to the first injection data in Table 7, and Std 3.0h refers to the sixth injection data in Table 7.

[0150] Table 7 System suitability results

[0151]

[0152] Table 8 Stability test results

[0153]

[0154] Conclusion: The system suitability results meet the requirements; the content of methyl bromoacetate in the reference solution and the test solution spiked with the test sample at 17.5 hours was between 80.0% and 120.0% compared with that at 0 hours, and the solution was stable.

[0155] 2.8.4 Accuracy and repeatability

[0156] Take the accuracy and repeatability solutions and inject one needle each. Record the chromatogram and peak area, and calculate the recovery rate and RSD. The system suitability results are the same as Table 7, the test results of the unspiked test solution are shown in Table 9, and the accuracy and repeatability results are shown in Table 10. Among them, A-spl M-3 refers to Sta-spl 7-hour data.

[0157] Table 9 Test results of unspiked test solution

[0158]

[0159] Table 10 Accuracy experimental results

[0160]

[0161] Conclusion: The system suitability results meet the requirements; the recoveries of methyl bromoacetate at three concentration levels were all in the range of 92.4% to 99.9%, and the RSD of the recoveries of 9 solutions was 2.6%, which all met the requirements.

[0162] 2.8.5 Limit of detection (LOD) and limit of quantification (LOQ)

[0163] The detection limit solution was injected into 3 needles, and the quantitation limit solution was injected into 6 needles to examine the peak area and signal-to-noise ratio. The system suitability results are shown in Table 11, the detection limit solution determination results are shown in Table 12, and the quantitation limit solution determination results are shown in Table 13.

[0164] Table 11 System suitability results

[0165]

[0166] Table 12 Test line experimental results

[0167]

[0168] Table 13 Quantitation limit experimental results

[0169]

[0170] Conclusion: The detection limit concentration is 10.69 ng / ml (equivalent to 10% of the limit concentration); the quantification limit concentration is 21.39 ng / ml (equivalent to 20% of the limit concentration), and the signal-to-noise ratio and peak area RSD both meet the requirements.

[0171] 2.8.6 Limitation and scope

[0172] Take each linear solution and inject 3 needles continuously, record the chromatogram and peak area. Among them, Lin-20% refers to the data of the three needles after LOQ, the system suitability results are the same as Table 11, the linear solution determination results are shown in Table 14, and the linear relationship diagram is shown in Figure 2 , Figure 2 It is a linear relationship graph.

[0173] Conclusion: The concentration of methyl bromoacetate derivatives has a good linear relationship with the peak area in the range of 21.39 ng / ml to 213.88 ng / ml. 2 It is 1.000, which meets the requirements.

[0174] Table 14 Linearity and range test results

[0175]

[0176]

[0177] Table 15. Summary of Methodological Validation

[0178]

[0179]

[0180] Example 3 Sample determination

[0181] 1. Reagents and reagents: acetonitrile, triethylamine, formic acid, 4-phenylthiophenol, ultrapure water.

[0182] 2. Instruments and appliances: Liquid chromatograph equipped with DAD or VWD detector, ultrapure water meter, ACE Excel 3C18-PFP (4.6×150mm, 3μm), electronic analytical balance, pipette, and standard volumetric flask.

[0183] 3. Preparation of stock solution:

[0184] 4-phenylthiophenol stock solution: Take about 25 mg of 4-phenylthiophenol, accurately weigh it and place it in a 25 ml volumetric flask, dissolve it in acetonitrile and dilute it to the mark, shake well to obtain;

[0185] Reference substance stock solution: take about 10 mg of methyl bromoacetate, accurately weigh it and place it in a 10 ml volumetric flask, dissolve it with acetonitrile and dilute it to the scale, shake well; take 100 μl of the above solution into a 10 ml volumetric flask, dilute it to the scale with acetonitrile, and shake well.

[0186] 4. Test solution: Weigh about 10 mg of compound 1 sample, accurately weigh, place in a 10 ml brown volumetric flask, accurately pipette 7.0 ml of acetonitrile to dissolve, then accurately pipette 50 μl of triethylamine and 250 μl of 4-phenylthiophenol stock solution into the same volumetric flask, make up to volume with acetonitrile, shake well, and react at room temperature for about 10 minutes. Prepare blank solution in the same way.

[0187] 5. Reference substance solution: Accurately pipette 7.0 ml of acetonitrile, 50 μl of triethylamine, 250 μl of 4-phenylthiophenol stock solution, and 100 μl of reference substance stock solution into the same 10 ml brown volumetric flask, make up to volume with acetonitrile, shake well, and react at room temperature for about 10 min.

[0188] 6. Sensitivity solution: Take about 10 mg of the reference substance of compound 1, accurately weigh it, put it in a 10 ml brown volumetric flask, accurately pipette 7.0 ml of acetonitrile to dissolve it, then accurately pipette 20 μl of the reference substance stock solution, 50 μl of triethylamine, and 250 μl of 4-phenylthiophenol stock solution into the same volumetric flask, make up to volume with acetonitrile, shake well, and react at room temperature for about 10 minutes.

[0189] 7. Operation method: Detect according to the general rules 0512 of the fourth part of the 2015 edition of the Chinese Pharmacopoeia. Use ACE EXCEL 3C18-PFP (4.6×150mm, 3μm) as the chromatographic column; use 0.1% formic acid solution as mobile phase A, acetonitrile as mobile phase B, the flow rate is 1ml per minute, and perform linear gradient elution according to the table below; the detection wavelength is 278nm; the column temperature is 45℃.

[0190]

[0191] Take 20μl of blank solvent (acetonitrile) and blank solution and inject them into the liquid chromatograph. There should be no interference. Take another 20μl of sensitivity solution and inject it into the liquid chromatograph. Record the chromatogram. The signal-to-noise ratio of the methyl bromoacetate derivative peak should be no less than 10, and the separation between it and the adjacent peak should be no less than 1.5. Then accurately measure 20μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph respectively, and record the chromatogram. If there is a methyl bromoacetate derivative peak in the test solution, calculate the content of bromoacetic acid by peak area according to the external standard method.

[0192] 8. Calculation

[0193] Content of methyl bromoacetate

[0194]

[0195] in:

[0196] A S = Peak area of ​​methyl bromoacetate derivative in the sample solution spectrum

[0197] A R = Average peak area of ​​methyl bromoacetate derivative peak in reference solution

[0198] C S = Sample concentration (mg / ml)

[0199] C R =Concentration of methyl bromoacetate in the reference solution (ng / ml)

[0200] 9. Limit: The content of methyl bromoacetate shall not exceed 50 ppm.

[0201] Table 3. Analytical methods for formic hydrazide detection in different batches of samples

[0202] Impurity Name limit Batch 1 Batch 2 Methyl bromoacetate 50ppm Not detected <20ppm

[0203] Conclusion: All validation results were within the acceptable range, indicating that the HPLC method was suitable for the detection of formic hydrazide in bulk drug compound 1 samples.

[0204] Comparative Example 4

[0205] The chromatographic conditions are consistent with those in Example 1, except that the elution gradient is as follows:

[0206] 0~20min, 60%A~50%A;

[0207] 20~30min, 50%A~5%A;

[0208] 30-40min, 5% A;

[0209] 40~40.1min, 5%~60%A;

[0210] 40.1~47min, 60%A.

[0211] The results are as follows Figure 3 , Figure 3 It is the chromatogram of comparative example 4. As shown, by Figure 3 It can be seen that the running time is long, which affects the inspection efficiency.

[0212] Comparative Example 5

[0213] 0~20min, 60%A~50%A;

[0214] 20~22min, 50%A~5%A;

[0215] 22-28min, 5% A;

[0216] 28~28.1min, 5%~60%A;

[0217] 28.1~35min, 60%A.

[0218] The results are as follows Figure 4 , Figure 4 It is the chromatogram of comparative example 5. As shown, by Figure 4 It can be seen that the baseline of the target peak is raised, which may affect the accuracy of the integration.

[0219] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting the genotoxic impurity methyl bromoacetate in the compound 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid, characterized in that: The steps include: A) reacting a compound, a solvent, triethylamine and a derivatizing agent to obtain a test solution; the compound is -((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid; B) reacting methyl bromoacetate, a solvent, triethylamine and a derivatization agent to obtain a reference substance solution; C) subjecting the test solution and the reference solution to high performance liquid chromatography, respectively, to obtain a test sample chromatogram and a reference sample chromatogram; The specific chromatographic parameters are as follows: Mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile; The elution procedure is: 0-7min, phase A 54%, phase B 46%; 7-11min: phase A 54-52%, phase B 46%-48%; 11-18min: Phase A 52-50%, Phase B 48-50%; 18-20min: 50% phase A, 50% phase B; 20-22min: Phase A 50%-5%, Phase B 50%-95%; 22-28min: Phase A 5%, Phase B 95%; 28-28.1min: Phase A 5%-54%, Phase B 95%-46%; 28.1-35min: phase A 54%, phase B 46%; D) calculating the content of methyl bromoacetate by external standard method according to the concentration of the reference substance, the peak area of ​​the reference substance solution in the chromatogram, and the peak area of ​​the component corresponding to the reference substance solution in the test solution, and based on the detection conditions in step C).

2. The detection method according to claim 1, characterized in that: The derivatization reagent is tetraphenylthiophenol.

3. The detection method according to claim 1, characterized in that: The reaction temperature of step A) is 25-35°C; the reaction time is 6-55min.

4. The detection method according to claim 1, characterized in that: Step A) The mass-volume ratio of the compound, solvent, triethylamine and derivatization reagent is (20 mg): 7 mL: (20-60 μL): (20 μL-50 μL).

5. The detection method according to claim 1, characterized in that: The chromatographic column is ACE EXCEL 3C18-PFP, and the specifications of the chromatographic column are 4.6×150 mm, 3 μm; the temperature of the chromatographic column is 45° C.

6. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 to 100 μL.

7. The detection method according to claim 1, characterized in that: The detection wavelength is 278 nm.

8. The detection method according to claim 1, characterized in that: The concentration of the methyl bromoacetate showed a good linear relationship in the range of 21.39 ng / ml-213.88 ng / ml.

9. The detection method according to claim 1, characterized in that: The detection limit concentration of the methyl bromoacetate is 10.69 ng / ml; the quantification limit concentration is 21.39 ng / ml.

10. The detection method according to claim 1, characterized in that: The solvent of step A) is acetonitrile; the solvent of step B) is acetonitrile; The reaction in step B) is specifically performed at 25-35° C. for 6-55 minutes.

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