Detection method of genotoxic impurity formylhydrazine

Abstract

The invention provides a method for detecting a genotoxic impurity formylhydrazine in a compound 2-((5-bromine-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, formic acid and a derivatization reagent to obtain a test solution, and detecting the genotoxic impurity formylhydrazine in the compound 2-((5-bromine-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1, 2, 4-triazole-3-yl)-sulfur) acetic acid. 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. Formylhydrazine, a solvent, formic acid 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 202311105065.8 and invention name “Method for detecting the genotoxic impurity formoylhydrazine”, 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 genotoxic impurity formoyl hydrazide. Background Art

[0003] Compound 1 is a targeted URAT1 inhibitor, with the chemical name 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid. URAT1 is an important renal anion transporter located on the brush border membrane of the epithelial cells of the renal tubules. It specifically transports uric acid from the renal tubules to the epithelial cells and is the main driving force for the reabsorption of uric acid in the renal tubules. Therefore, if the urate transporter URAT1 can be significantly inhibited, the excretion of uric acid in the body will increase, thereby reducing blood uric acid levels and reducing the possibility of gout attacks. Prior art 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, compared with the use of xanthine oxidase inhibitors alone, the additive effect of the combined use is not very significant. At the same time, the 400 mg / day dose of Lesinurad was not approved because of the significantly increased toxic side effects observed at high doses (the incidence of kidney-related adverse events, especially kidney stones, is high), although the combination of drugs at high doses showed a higher additive effect. Therefore, the FDA requires Lesinurad labels to add a black box warning to alert medical staff to the risk of acute renal failure caused by Lesinurad, especially when not used in combination with XOI. If Lesinurad is used at an approved dose, the risk of renal failure is higher. At the same time, the FDA requires AstraZeneca to continue to investigate renal and cardiovascular safety after Lesinurad is launched. For long-term medication for a metabolic disease, drug safety is particularly important.

[0006] Therefore, it is very necessary to develop a detection method for the genotoxic impurity formoylhydrazide in order to accurately reflect the content of the genotoxic impurity formoylhydrazide in the raw material or preparation of compound 1 and provide a reasonable basis for the formulation of quality standards. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting the genotoxic impurity formoyl hydrazide. 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 formoyl hydrazide. 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 genotoxic impurity formic acid hydrazide 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 it comprises the following steps:

[0014] A) reacting a compound, a solvent, formic acid 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 formic hydrazide, a solvent, formic acid and a derivatizing 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% phosphoric acid solution, mobile phase B: acetonitrile;

[0019] The elution procedure is:

[0020] 0-11min, phase A 80%-55%, phase B 20%-45%;

[0021] 11-15min: Phase A 55-20%, Phase B 45%-80%;

[0022] 15-20min: Phase A 20%, Phase B 80%;

[0023] 20~20.1min Phase A 20%~80%, Phase B 80%~20%;

[0024] 20.1~27min Phase A 80%, Phase B 20%;

[0025] D) calculating the content of formic hydrazide by an 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 in the chromatogram, and based on the detection conditions in step C).

[0026] The present invention provides a method for detecting the genotoxic impurity formoyl hydrazide 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, formic acid and a derivatization reagent to obtain a test solution.

[0027] According to the present invention, after the compound is dissolved in a solvent, formic acid and a derivatization reagent are added and mixed, and the mixture is vortexed and reacted to obtain a test solution.

[0028] 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.

[0029] The structure is shown in formula (I):

[0030]

[0031] In some preferred embodiments of the present invention, the derivatization agent is selected from one or more of benzaldehyde, p-methylbenzaldehyde or p-methoxybenzaldehyde.

[0032] In some preferred embodiments of the present invention, the derivatization agent is p-methoxybenzaldehyde.

[0033] The inventors creatively discovered that when p-methoxybenzaldehyde is used as a derivatization reagent, the impurities in the derivatization reagent have minimal interference with the target derivatization peak.

[0034] The solvent of the present invention includes but is not limited to acetonitrile.

[0035] In some preferred embodiments of the present invention, the reaction temperature is 50-70° C. and the reaction time is 30-90 min.

[0036] In some preferred embodiments of the present invention, the reaction temperature is 55-65° C. and the reaction time is 40-80 min.

[0037] In some preferred embodiments of the present invention, the reaction temperature is 60° C. and the reaction time is 60 min.

[0038] The present inventors creatively discovered that within the above derivatization temperature and time, the derivatization result is good.

[0039] In some preferred embodiments of the present invention, the mass-volume ratio of the compound, solvent, formic acid and derivatization reagent is (20 mg): 7 mL: (150-250 μL): (5.6 mg-11.4 mg).

[0040] In some preferred embodiments of the present invention, the mass-volume ratio of the compound, solvent, formic acid and derivatization reagent is (20 mg): 7 mL: (170-220 μL): (6.4-10 mg).

[0041] In some preferred embodiments of the present invention, the mass-volume ratio of the compound, solvent, formic acid and derivatization reagent is (20 mg): 7 mL: 200 μL: 9 mg.

[0042] 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.

[0043] The formic hydrazide, solvent, formic acid and a derivatization reagent are reacted to obtain a reference solution.

[0044] The solvent of the present invention includes but is not limited to acetonitrile.

[0045] According to the present invention, the above reaction is preferably vortexed and reacted at 50-70°C for 30-90 minutes; more preferably vortexed and reacted at 55-65°C for 40-80 minutes; most preferably vortexed and reacted at 60°C for 60 minutes.

[0046] The experimental results show that the concentration of the formic acid hydrazide of the present invention is in a good linear relationship between 30.62 ng / ml and 408.32 ng / ml.

[0047] The present invention also includes the preparation of the system adaptability solution, which is specifically: adding the compound into a solvent to dissolve it, then adding formic acid, mixing the derivatization reagent and the reference substance, and reacting to obtain the system adaptability solution.

[0048] 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 reference sample concentration, 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 formoyl hydrazide is calculated by an external standard method.

[0049] The present invention does not limit the above-mentioned external standard method for calculating the content of formic hydrazide, which is well known to those skilled in the art.

[0050] The specific chromatographic parameters of the present invention are as follows:

[0051] Mobile phase A: 0.1% phosphoric acid solution, mobile phase B: acetonitrile;

[0052] The elution procedure is:

[0053] 0-11min, phase A 80%-55%, phase B 20%-45%;

[0054] 11-15min: Phase A 55-20%, Phase B 45%-80%;

[0055] 15-20min: Phase A 20%, Phase B 80%;

[0056] 20~20.1min Phase A 20%~80%, Phase B 80%~20%;

[0057] 20.1~27min Phase A 80%, Phase B 20%;

[0058] The present invention achieves good baseline separation, good separation of each peak and stable baseline by selecting the mobile phase gradient.

[0059] In the present invention, the chromatographic column is INERTSIL ODS-4, the specifications of the chromatographic column are 4.6×250 mm, 5 μm; and the temperature of the chromatographic column is 50° C.

[0060] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above 50°C condition.

[0061] In the present invention, the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 μL.

[0062] 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.

[0063] In the present invention, the detection wavelength is 286 nm.

[0064] In the present invention, the detection limit concentration of formic hydrazide is 15.31 ng / mL; the quantification limit concentration is 30.62 ng / mL.

[0065] The invention has low detection limit, low quantitative limit, high separation degree and good effect.

[0066] The present invention provides a method for detecting genotoxic impurity formoyl hydrazide 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, formic acid and a derivatization reagent to obtain a test solution; the compound is -((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)- sulfur) acetic acid; B) reacting formohydrazide, solvent, formic acid and derivatization reagent to obtain reference solution; C) subjecting the test solution and the reference solution to high performance liquid chromatography detection, respectively, to obtain a test chromatogram and a reference chromatogram; D) according to the reference concentration, 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 in the chromatogram, and based on the detection conditions in step C), calculating the content of formohydrazide by external standard method. 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

[0067] Figure 1 Benzaldehyde is used as the derivatization reagent;

[0068] Figure 2 p-Tolualdehyde is used as the derivatization reagent;

[0069] Figure 3 p-Anisaldehyde is used as the derivatization reagent;

[0070] Figure 4 is specificity-blank solvent;

[0071] Figure 5 is the specificity-blank derivatization solution;

[0072] Figure 6 It is a specificity-reference substance derivatization solution;

[0073] Figure 7 It is a specificity-test product derived solution;

[0074] Figure 8 For specificity - the test sample is spiked with the derivatization solution;

[0075] Fig. 9 is a linear relationship graph;

[0076] Fig.10 It is the quantitative limit solution;

[0077] Fig.11 is the detection limit solution;

[0078] Fig.12 is the recovery rate - spiked with 50% solution;

[0079] Fig.13 is the recovery rate - spiked 100% solution;

[0080] Fig.14 The recovery is the 150% spike solution. DETAILED DESCRIPTION

[0081] The present invention provides a method for detecting a genotoxic impurity, formoyl hydrazide. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0082] To further illustrate the present invention, a method for detecting a genotoxic impurity, formic acid hydrazide, provided by the present invention is described in detail below in conjunction with examples.

[0083] The batch number of the formic acid hydrazide of the present invention is 20171224 and the source is Beijing Coupling Technology Co., Ltd.

[0084] Compound 1, batch number H21715-31-P1, was obtained from WuXi AppTec.

[0085] Example 1 Detection method of genotoxic formic acid hydrazide impurity

[0086] The preparation process of compound 1 (see WO2021249468A1 for details) uses hydrazide, which has a risk of genotoxicity. In order to ensure the safety of the finished product, a sensitive analytical method is developed to detect its residues.

[0087] The ultraviolet absorption of formate hydrazide is weak, and it is difficult to directly measure it using HPLC or GC methods. Therefore, a derivatization method is used to measure formate hydrazide derivatives with strong ultraviolet absorption, thereby determining the content of formate hydrazide.

[0088] The analytical method for formoyl hydrazide detection was finally determined by examining different types and amounts of derivatization reagents, formic acid dosage, reaction temperature and reaction time, optimizing elution conditions, column temperature, etc., and examining the separation between the derivatization reagent and adjacent impurities. The chromatographic conditions are shown in Table 1.

[0089] Table 1. Chromatographic conditions

[0090]

[0091]

[0092] (I) Screening of derivatization reaction conditions

[0093] (1) Preparation of stock solution

[0094] Different types of benzaldehyde stock solutions: Take 500 mg of each benzaldehyde into a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and prepare a concentration of 20 mg / ml.

[0095] Formic hydrazide stock solution ①: Take 25 mg of formic hydrazide into a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as formic hydrazide stock solution ① with a concentration of 1.0 mg / ml.

[0096] Formic hydrazide stock solution ②: Measure formic hydrazide stock solution ① into a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as formic hydrazide stock solution ② with a concentration of 10 μg / ml.

[0097] (2) Screening of derivatization reagents

[0098] The derivatization reaction effects of the derivatization reagents benzaldehyde, p-methylbenzaldehyde, and p-methoxybenzaldehyde were investigated respectively. The results are as follows Figure 1 to Figure 3 As shown, Figure 1 Benzaldehyde is used as the derivatization reagent; Figure 2 p-Tolualdehyde is used as the derivatization reagent. Figure 3 p-Anisaldehyde is used as the derivatization reagent.

[0099] The results showed that the impurities in the derivatization reagent of p-methoxybenzaldehyde had the least interference on the target derivatization peak, so p-methoxybenzaldehyde was selected as the derivatization reagent.

[0100] (3) Investigation of formic acid dosage

[0101] Preparation of reference derivatization solutions with different formic acid dosages: Pipette 7 ml of acetonitrile, different amounts of formic acid, 570 μl of p-methoxybenzaldehyde stock solution, and 200 μl of formic hydrazide stock solution② into the same 10 ml volumetric flask, make up to volume with acetonitrile, vortex for 30 seconds, and react in a 60°C water bath for 60 minutes.

[0102] Table 2

[0103]

[0104]

[0105] The experimental results show that the use of different amounts of formic acid has no significant effect on the target peak area. Therefore, there is no need to further adjust the amount of formic acid, and the amount of formic acid is initially set at 200 μl.

[0106] (4) Investigation of reaction temperature

[0107] Preparation of reference substance derivatization solution at different reaction temperatures: Pipette 7 ml of acetonitrile, 200 μl, 570 μl of p-methoxybenzaldehyde stock solution, and 200 μl of formate hydrazide stock solution② into the same 10 ml volumetric flask, make up to volume with acetonitrile, vortex for 30 seconds, and react in a water bath at different temperatures for 60 minutes.

[0108] Table 3

[0109] Different temperature (℃) Target peak area 50 30807 60 30527 70 31236

[0110] The results showed that adjusting the reaction temperature had no effect on the area of ​​the derived target peak. The reaction temperature was no longer adjusted to avoid differences in reaction conditions caused by subsequent reaction temperature fluctuations. Therefore, the reaction temperature was set at 60°C.

[0111] (5) Investigation of the amount of derivatization reagent used

[0112] Preparation of reference solutions with different amounts of derivatization reagents: Pipette 7 ml of acetonitrile, 200 μl of formic acid, different amounts of p-methoxybenzaldehyde stock solution, and 200 μl of formoyl hydrazide stock solution② into the same 10 ml volumetric flask, make up to volume with acetonitrile, vortex for 30 seconds, and react in a 60°C water bath for 60 minutes.

[0113] Table 4

[0114] Different derivatization reagent dosage Target peak area 280μL 31083 450μL 30983 570μL 30938

[0115] The results showed that adjusting the amount of derivatization reagent had no significant effect on the peak area of ​​the derivatized target. Taking into account the need for a linear limit concentration of 200%, the amount of derivatization reagent was selected to be 450 μl.

[0116] (6) Investigation of reaction time

[0117] Based on the optimization of different conditions (1) to (5), the derivatization reaction time was investigated. The derivatization reaction results of 100% and 200% limit concentration reference solution at different reaction times are shown in the table below.

[0118] Table 5

[0119]

[0120] The results showed that as the reaction time increased, the increase in the target peak was not significant, so the reaction time was set at 60 minutes.

[0121] Based on the results of investigations under different conditions (1) to (6), the derivatization reaction conditions were determined as follows: 200 μl of formic acid, 60°C of reaction temperature, 570 μl of derivatization reagent, and 60 min of reaction time; and the amount of derivatization reagent can be adjusted accordingly according to the concentration of the derivatization reagent stock solution.

[0122] Example 2

[0123] 2.1 Preparation of stock solution:

[0124] p-Anisaldehyde stock solution-1: Take 0.2579 g of p-Anisaldehyde, accurately weigh it into a 25 ml brown volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well;

[0125] p-Anisaldehyde stock solution-2: Take 0.2514 g of p-Anisaldehyde, accurately weigh it into a 25 ml brown volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well;

[0126] Prepare stock solution ① with a concentration of 1.0446 mg / mL, stock solution ② with a concentration of 10.4464 μg / mL, stock solution ③ with a concentration of 1.0208 mg / mL, and stock solution ④ with a concentration of 10.208 μg / mL respectively.

[0127] 2.2 Configuration of proprietary solution:

[0128] Blank: acetonitrile solution;

[0129] Sys-1: Accurately pipette 7 mL of acetonitrile, 200 μL of formic acid, and 1 lml of p-methoxybenzaldehyde stock solution into the same 10 ml brown volumetric flask, add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain the product.

[0130] Sys-2: Accurately pipette 7 mL of acetonitrile, 200 μL of formic acid, 1 ml of p-anisaldehyde stock solution, and 200 μL of p-anisaldehyde stock solution 2 into the same 10 mL brown volumetric flask. Add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain Sys-2.

[0131] Sys-3: Accurately weigh 20.07 mg of compound 1 into a 10 mL brown volumetric flask, transfer 7 mL of acetonitrile to dissolve it, add 200 μL of formic acid and 1 l mL of p-methoxybenzaldehyde stock solution into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60 ° C for 60 min to obtain Sys-3.

[0132] Sys-4: Accurately weigh 20.29 mg of compound 1 into a 10 mL brown volumetric flask, transfer 7 mL of acetonitrile to dissolve it, add 200 μL of formic acid, 1 ml of p-anisaldehyde stock solution, and 200 μL of p-anisaldehyde stock solution 2 into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60 °C for 60 min to obtain Sys-4.

[0133] 2.3 Preparation of stability solution

[0134] Blank: acetonitrile solution;

[0135] Std: Accurately pipette 7 mL of acetonitrile, 200 μL of formic acid, 1 ml of p-anisaldehyde stock solution, and 200 μL of p-anisaldehyde stock solution 2 into the same 10 mL brown volumetric flask. Add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain the product.

[0136] Sta-spl: accurately weigh 20.29 mg of compound 1 into a 10 mL brown volumetric flask, transfer 7 mL of acetonitrile to dissolve it, add 200 μL of formic acid, 11 mL of p-anisaldehyde stock solution, and 200 μL of p-anisaldehyde stock solution 2 into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60 °C for 60 minutes to obtain the product.

[0137] 2.4 Configuration of precision solution:

[0138] Blank: acetonitrile solution;

[0139] Std: Accurately pipette 7 mL of acetonitrile, 200 μL of formic acid, 1 ml of p-anisaldehyde stock solution, and 200 μL of p-anisaldehyde stock solution 2 into the same 10 mL brown volumetric flask. Add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain the product.

[0140] 2.5 Accuracy solution configuration:

[0141] Blank: acetonitrile solution.

[0142] Std: Same as Sys-2 under the specificity test.

[0143] Preparation of unsampled solution: Take about 20 mg of compound 1, weigh accurately, put it into a 10 ml brown volumetric bottle, accurately pipette 7 ml of acetonitrile to dissolve it, then accurately pipette 200 μL of formic acid and 1 ml of p-methoxybenzaldehyde stock solution-1 into the same volumetric bottle, add acetonitrile to make up the volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain. Repeat the preparation for 2 times.

[0144] Sample addition - low concentration: Take about 20 mg of compound 1, weigh accurately, put it into a 10 ml brown volumetric flask, accurately pipette 7 ml of acetonitrile to dissolve it, then accurately pipette 200 μL of formic acid, 1 ml of p-methoxybenzaldehyde stock solution-1, and 100 μL of formic hydrazide stock solution ② into the same volumetric flask, add acetonitrile to the volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain. Repeat the preparation for 3 times.

[0145] Sample addition - medium concentration: Take about 20 mg of compound 1, weigh accurately, put it into a 10 ml brown volumetric bottle, accurately pipette 7 ml of acetonitrile to dissolve it, then accurately pipette 200 μL of formic acid, 1 ml of p-methoxybenzaldehyde stock solution-1, and 200 μL of formic hydrazide stock solution ② into the same volumetric bottle, add acetonitrile to make up the volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain. Repeat the preparation for 3 times.

[0146] Sample addition - high concentration: Take about 20 mg of compound 1, weigh accurately, put it into a 10 ml brown volumetric bottle, accurately pipette 7 mL of acetonitrile to dissolve it, then accurately pipette 200 μL of formic acid, 1 mL of p-methoxybenzaldehyde stock solution-1, and 300 μL of formic hydrazide stock solution ② into the same volumetric bottle, add acetonitrile to make up the volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain. Repeat the preparation for 3 times.

[0147] 2.6 Detection limit and quantification limit solution configuration:

[0148] Detection limit solution: Accurately pipette 7 mL of acetonitrile, 200 μL of formic acid, 1 mL of p-methoxybenzaldehyde stock solution-2, and 15 μL of formoyl hydrazide stock solution④ into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain the solution.

[0149] Quantitation limit solution: Accurately pipette 7 mL of acetonitrile, 200 μL of formic acid, 1 mL of p-methoxybenzaldehyde stock solution-2, and 30 μL of formic hydrazide stock solution④ into the same 10 mL brown volumetric flask, add acetonitrile to make up to volume, vortex for about 30 seconds, and react at 60°C for 60 minutes to obtain the solution.

[0150] 2.7 Linear solution configuration:

[0151] Prepare formic acid hydrazide solutions with concentrations of 30.6241 to 408.3216 ng / mL respectively in the same manner as above;

[0152] 2.8 Use the chromatographic conditions in Table 1 to verify the methodology.

[0153] 2.8.1 Exclusivity

[0154] 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, where: Sys-4 refers to Sta-spl 0 hour data. The specificity overlay diagram is shown in Figure 4 , Figure 4 This is a specificity overlay map, from top to bottom: sys1, sys2, sys3, sys4. Figure 6 It is a specificity-reference substance derivatization solution; Figure 7 It is a specificity-test product derived solution; Figure 8 For specificity - the test sample is spiked with the derivatization solution;

[0155] Table 6-1

[0156]

[0157] Conclusion: The blank solvent does not interfere with the target chromatographic peak; the blank derivative solution has no interference at the target chromatographic peak and the minimum separation between the target chromatographic peak and nearby impurity peaks is 2.0, and the method has good specificity.

[0158] 2.8.2 Injection precision

[0159] Take the precision solution and inject it 6 times continuously, and record the chromatogram. The results of the precision solution injection are shown in Table 6-2.

[0160] Table 6-2 Injection precision results

[0161]

[0162] Conclusion: The RSD of the target peak area after 6 consecutive injections of the solution was 0.6%, and the injection precision was good.

[0163] 2.8.3 Solution stability

[0164] The stability solution stored at room temperature was sampled and the changes in the content of formate hydrazide in the reference solution and the spiked test solution within 13.5 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 2.5h refers to the sixth injection data in Table 7.

[0165] Table 7 System suitability results

[0166]

[0167] Table 8 Stability test results

[0168]

[0169] Conclusion: The system suitability results meet the requirements; the content of formate hydrazide in the reference solution and the test solution spiked with the test sample at 13.5 hours was between 80.0% and 120.0% compared with that at 0 hours, and the solution was stable.

[0170] 2.8.4 Accuracy and repeatability

[0171] 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.

[0172] Fig.12 is the recovery rate - spiked with 50% solution; Fig.13 is the recovery rate - spiked 100% solution; Fig.14 The recovery is the 150% spike solution.

[0173] Table 9 Test results of unspiked test solution

[0174]

[0175] Table 10 Accuracy experimental results

[0176]

[0177] Conclusion: The system suitability results meet the requirements; the recoveries of formate hydrazide content at three concentration levels were all in the range of 95.0% to 101.0%, and the RSD of the recoveries of 9 solutions was 1.8%, which all met the requirements.

[0178] 2.8.5 Limit of detection (LOD) and limit of quantification (LOQ)

[0179] Take the detection limit solution and inject it into 3 needles, take the quantitation limit solution and inject it into 6 needles, and record the chromatogram, 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. Fig.10 It is the quantitative limit solution; Fig.11 The detection limit solution.

[0180] Table 11 System suitability results

[0181]

[0182] Table 12 Test line experimental results

[0183]

[0184] Table 13 Quantitation limit experimental results

[0185]

[0186] Conclusion: The detection limit concentration is 15.31 ng / ml (equivalent to 7.5% of the limit concentration); the quantification limit concentration is 30.62 ng / ml (equivalent to 15% of the limit concentration), and the signal-to-noise ratio and peak area RSD both meet the requirements.

[0187] 2.8.6 Limitation and scope

[0188] Take each linear solution and inject 3 needles continuously, record the chromatogram and peak area. Among them, Lin-15% 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 Fig. 9 , Fig. 9 It is a linear relationship graph.

[0189] Conclusion: The concentration of formoyl hydrazide derivatives has a good linear relationship with the peak area in the range of 30.62ng / ml to 408.32ng / ml. 2 It is 1.000, which meets the requirements.

[0190] Table 14 Linearity and range test results

[0191]

[0192] Table 15. Summary of Methodological Validation

[0193]

[0194]

[0195] Example 3 Sample determination

[0196] 1. Reagents and reagents: acetonitrile, phosphoric acid, formic acid, p-anisaldehyde, ultrapure water.

[0197] 2. Instruments and appliances: Liquid chromatograph equipped with DAD or VWD detector, ultrapure water analyzer, INERTSIL ODS-4 (4.6×250mm, 5μm), electronic analytical balance, vortex vortexer, pipette, standard volumetric flask, and water bath.

[0198] 3. Preparation of stock solution:

[0199] p-Anisaldehyde stock solution: Take about 250 mg of p-Anisaldehyde, accurately weigh it and place it in a 25 ml brown volumetric bottle, dissolve it in acetonitrile and dilute it to the scale, shake well and you are done;

[0200] Reference substance stock solution: take about 10 mg of formic hydrazide, accurately weigh and place in a 10 ml brown volumetric flask, dissolve with acetonitrile and dilute to the mark, shake well; take 100 μl of the above solution into a 10 ml brown volumetric flask, dilute with acetonitrile to the mark, shake well.

[0201] 4. Test solution: Weigh about 20 mg of compound 1 sample (prepared according to WO2021249468A1 method), accurately weigh, and place in a 10 ml brown volumetric flask. Accurately pipette 7.0 ml of acetonitrile to dissolve, then accurately pipette 200 μl of formic acid and 1.0 ml of p-methoxybenzaldehyde stock solution into the same volumetric flask, dilute to scale with acetonitrile, vortex for 30 seconds, and react at 60 ° C for about 60 minutes. Prepare a blank solution in the same way.

[0202] 5. Reference substance solution: Accurately pipette 7.0 ml of acetonitrile, 200 μl of formic acid, 1.0 ml of p-methoxybenzaldehyde stock solution and 200 μl of reference substance stock solution into the same 10 ml brown volumetric flask, make up to the mark with acetonitrile, vortex for 30 seconds, and react at 60°C for about 60 minutes.

[0203] 6. System suitability solution: Take about 20 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 200 μl of formic acid, 1.0 ml of p-methoxybenzaldehyde stock solution and 30 μl of reference substance stock solution into the same volumetric flask, make up to the scale with acetonitrile, vortex for 30 seconds, and react at 60°C for about 60 minutes.

[0204] 7. Operation method: Detect according to the general rule 0512 of the fourth part of the 2015 edition of the Chinese Pharmacopoeia. Use INERTSIL ODS-4 (4.6×250mm, 5μm) as the chromatographic column; use 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, the flow rate is 1ml per minute, and linear gradient elution is performed according to the table below; the detection wavelength is 286nm; the column temperature is 50℃.

[0205]

[0206] Take 10μl of blank solvent (acetonitrile) and blank solution and inject them into the liquid chromatograph. There should be no interference. Take another 10μl of system suitability solution and inject it into the liquid chromatograph. Record the chromatogram. The signal-to-noise ratio of the formoyl hydrazide 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 10μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph respectively, and record the chromatogram. If there is a formoyl hydrazide derivative peak in the test solution, calculate the formoyl hydrazide content by peak area according to the external standard method.

[0207] 8. Calculation

[0208] Formic acid hydrazide content

[0209]

[0210] in:

[0211] A S = Peak area of ​​formic acid hydrazide derivative in the sample solution spectrum

[0212] A R = Average peak area of ​​formate hydrazide derivative peak in reference solution

[0213] C S = Sample concentration (mg / ml)

[0214] C R =Concentration of formic hydrazide in reference solution (ng / ml)

[0215] 9. Limit: The content of formate hydrazide shall not exceed 100 ppm.

[0216] Table 3. Analytical methods for formic hydrazide detection in different batches of samples

[0217] Impurity Name limit Batch 1 Batch 2 Formic hydrazide 100ppm Not detected Not detected

[0218] Conclusion: All validation results were within the acceptable range, indicating that the HPLC method was suitable for the detection of formoyl hydrazide in bulk drug compound 1 samples.

[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 formoyl hydrazide 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, formic acid 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 formic hydrazide, solvent, formic acid and a derivatizing 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% phosphoric acid solution, mobile phase B: acetonitrile; The elution procedure is: 0-11min, phase A 80%-55%, phase B 20%-45%; 11-15min: Phase A 55-20%, Phase B 45%-80%; 15-20min: Phase A 20%, Phase B 80%; 20~20.1min Phase A 20%~80%, Phase B 80%~20%; 20.1~27min Phase A 80%, Phase B 20%; D) calculating the content of formic hydrazide by an 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 in the chromatogram, and based on the detection conditions in step C).

2. The detection method according to claim 1, characterized in that: The derivatization agent is selected from one or more of benzaldehyde, p-methylbenzaldehyde or p-methoxybenzaldehyde.

3. The detection method according to claim 1, characterized in that: Step A) The reaction temperature is 50-70°C; the reaction time is 30-90 min.

4. The detection method according to claim 1, characterized in that: Step A) The mass-volume ratio of the compound, solvent, formic acid and derivatization reagent is (10-100 mg): 7 mL: (100-300 μL): (5-20 mg).

5. The detection method according to claim 1, characterized in that: The chromatographic column is INERTSIL ODS-4, and the specifications of the chromatographic column are 4.6×250 mm, 5 μm; the temperature of the chromatographic column is 50-60° C.

6. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.5-2.0 mL / min; the injection volume is 5-50 μL.

7. The detection method according to claim 1, characterized in that: The detection wavelength is 284-290 nm.

8. The detection method according to claim 1, characterized in that: The concentration of the formic hydrazide is in a good linear relationship between 30.62 ng / ml and 408.32 ng / ml.

9. The detection method according to claim 1, characterized in that: The detection limit concentration of the formic acid hydrazide is 15.31 ng / mL; the quantification limit concentration is 30.62 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 step B) reaction is specifically as follows: after vortexing, react at 50-70° C. for 30-90 minutes.

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