Detection method of genotoxic impurity bromoacetic acid

Abstract

The invention provides a bromoacetic acid detection method which comprises the following steps: A) reacting a compound, a solvent, alkali and a derivatization reagent to obtain a test solution; reacting bromoacetic acid, a solvent, alkali and a derivatization reagent to obtain a reference solution; b) respectively performing high performance liquid chromatography detection on the test solution and the reference solution to obtain a test chromatogram and a reference chromatogram; and C) calculating the content of bromoacetic acid 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 a component, corresponding to the reference substance solution, in the test solution in the chromatogram. The method provided by the invention is good in specificity, good in durability, low in detection limit, low in quantification limit, high in separation degree and relatively accurate in result. 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 202311106838.4 and invention name “Method for detecting genotoxic impurity bromoacetic acid”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the technical field of drug quality detection, and in particular to a method for detecting bromoacetic acid, a genotoxic impurity. 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 epithelial cells in 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 bromoacetic acid in order to accurately reflect the content of the genotoxic impurity bromoacetic acid in the raw materials or preparations 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 bromoacetic acid. 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 bromoacetic acid. 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 bromoacetic acid, 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, comprising the following steps:

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

[0015] reacting bromoacetic acid, a solvent, a base and a derivatization agent to obtain a reference substance solution;

[0016] B) 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 chromatographic parameters are as follows:

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

[0019] The elution procedure is:

[0020] 0-22min, phase A 60%, phase B 40%;

[0021] 22-23min: Phase A 60%-5%, Phase B 40%-95%;

[0022] 23-30min: Phase A 5%, Phase B 95%;

[0023] 30~30.1min Phase A 5%~60%, Phase B 95%~40%;

[0024] 30.1~38min Phase A 60%, Phase B 40%;

[0025] Alternatively, the elution program is:

[0026] 0-20min, phase A 60%, phase B 40%;

[0027] 20-22min: Phase A 60%-5%, Phase B 40%-95%;

[0028] 22-32min: Phase A 5%, Phase B 95%;

[0029] 32~32.1min Phase A 5%~60%, Phase B 95%~40%;

[0030] 32.1~38min Phase A 60%, Phase B 40%;

[0031] C) calculating the content of bromoacetic acid 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 in the chromatogram.

[0032] The ultraviolet absorption of bromoacetic acid is weak, and it is difficult to directly measure it using HPLC or GC methods. Therefore, the present application uses a derivatization method to measure bromoacetic acid derivatives with strong ultraviolet absorption, thereby determining the content of bromoacetic acid.

[0033] The present invention first prepares the test solution and the reference solution as follows:

[0034] The compound, solvent, base and derivatization reagent are reacted to obtain a test solution; the compound is 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid;

[0035] Bromoacetic acid, a solvent, a base and a derivatization reagent are reacted to obtain a reference solution.

[0036] In the present application, the object to be tested is 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid, the structure of which is shown in formula (I):

[0037]

[0038] The present invention firstly adopts a solvent to dissolve a derivatization reagent to prepare a 1 mg / mL derivatization reagent solution; then adopts a solvent to dissolve a test object, and then mixes with a base and the derivatization reagent solution for reaction to obtain a test solution.

[0039] In some possible implementations, the derivatization reagent is selected from 4-phenylthiophenol, the base is selected from triethylamine, and the solvent is selected from acetonitrile. The inventors creatively discovered that when 4-phenylthiophenol is used as the derivatization reagent, the impurities in the derivatization reagent have the least interference with the target derivatization peak.

[0040] In some possible implementations, the mass-volume ratio of the compound, solvent, base and derivatization agent is 10 mg: 7-15 mL: 50-100 μL, 100-400 μg, preferably 10 mg: 8-10 mL: 60-80 μL, 200-300 μg, and more preferably 10 mg: 9 mL: 70 μL, 250 μg. Within the above ratio range, the derivatization of the present invention is complete, the peak area has no significant difference, and the target peak separation is good.

[0041] In some possible implementations, the reaction is specifically: vortexing the mixture and then reacting, the reaction temperature is 15 to 35°C, preferably room temperature; the reaction time is 20 to 40 minutes, preferably 30 minutes. In some possible implementations, after the reaction is completed, formic acid is used to adjust the pH value, and the volume mass ratio of formic acid to the compound is preferably 70 to 120 μL: 10 mg, more preferably 90 μL: 10 mg. At the above derivatization temperature and time, the derivatization result is good.

[0042] In some possible implementations, the present application uses a solvent to dissolve the reference bromoacetic acid and the derivatization reagent, respectively, to obtain a 1 mg / mL bromoacetic acid solution and a 1 mg / mL derivatization reagent solution, respectively, and then the solvent, base, derivatization reagent solution and bromoacetic acid solution are mixed and reacted to obtain a reference solution. Similar to the preparation of the test solution, during the preparation of the reference solution, the derivatization reagent is selected from 4-phenylthiophenol, the base is selected from triethylamine, and the solvent is selected from acetonitrile. In some possible implementations, the mass-volume ratio of the bromoacetic acid, solvent, base and derivatization reagent is 100 μg: 7-15 mL: 50-100 μL, 100-400 μg, preferably 100 μg: 8-10 mL: 60-80 μL, 200-300 μg, and more preferably 100 μg: 9 mL: 70 μL, 250 μg. In some possible implementations, the reaction is specifically: vortexing the mixture and then reacting, the reaction temperature is 15-35°C, preferably room temperature, and the reaction time is 20-40min, preferably 30min. In some possible implementations, after the reaction is completed, formic acid is used to adjust the pH value, and the volume mass ratio of formic acid to bromoacetic acid is preferably 70-120μL:100μg, more preferably 90μL:100μg.

[0043] After obtaining the test solution and the reference solution, they are respectively subjected to high performance liquid chromatography to obtain a test sample chromatogram and a reference sample chromatogram;

[0044] The chromatographic parameters are as follows:

[0045] Mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile;

[0046] The elution procedure is:

[0047] 0-22min, phase A 60%, phase B 40%;

[0048] 22-23min: Phase A 60%-5%, Phase B 40%-95%;

[0049] 23-30min: Phase A 5%, Phase B 95%;

[0050] 30~30.1min Phase A 5%~60%, Phase B 95%~40%;

[0051] 30.1~38min Phase A 60%, Phase B 40%;

[0052] Alternatively, the elution program is:

[0053] 0-20min, phase A 60%, phase B 40%;

[0054] 20-22min: Phase A 60%-5%, Phase B 40%-95%;

[0055] 22-32min: Phase A 5%, Phase B 95%;

[0056] 32~32.1min Phase A 5%~60%, Phase B 95%~40%;

[0057] 32.1~38min Phase A 60%, Phase B 40%.

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

[0059] In some possible implementations, the chromatographic column is Luna 5u Phenyl-Hexyl, the chromatographic column specifications are 4.6×250 mm, 5 μm, and the chromatographic column temperature is 40° C. The chromatographic peaks of the chromatographic column of the present invention are symmetrical and have good separation at 40° C.

[0060] In some possible implementations, the mobile phase flow rate is 1.0 mL / min and the injection volume is 20 μL. The present invention found that the chromatographic peaks were better separated and the peak shape was more symmetrical at a flow rate of 1 ml / min, which is the most preferred solution.

[0061] In some possible implementations, the detection wavelength is 278 nm.

[0062] After obtaining the test sample chromatogram and the reference sample chromatogram, the content of bromoacetic acid is calculated by the external standard method 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. The present invention does not limit the method for calculating the content of bromoacetic acid by the external standard method, and a method well known to those skilled in the art can be used.

[0063] In some possible implementations, the detection method provided in the present application further includes: before detecting the test solution, using a sensitivity solution for detection. The sensitivity solution is prepared according to the following method: after dissolving the compound, mixing and reacting with a bromoacetic acid reference substance, a base and a derivatization reagent. A typical preparation method of the sensitivity solution is as follows:

[0064] Take 10 mg of the reference substance of the compound, put it in a 10 ml volumetric flask, take 9.0 ml of acetonitrile to dissolve it, then take 20 μl of the reference substance stock solution, 70 μl of triethylamine, and 250 μl of 4-phenylthiophenol stock solution into the same volumetric flask, vortex and react at room temperature for about 30 minutes, add 90 μl of formic acid, make up to volume with acetonitrile, and mix well.

[0065] When using sensitive solution for detection, the chromatogram should meet the following requirements:

[0066] The signal-to-noise ratio of the bromoacetic acid derivative peak should be no less than 10, and the separation between it and the adjacent peaks should be no less than 1.5.

[0067] The experimental results show that, in the detection method provided by the present invention, the concentration of bromoacetic acid has a good linear relationship in the range of 20.77 ng / ml to 207.74 ng / ml; the detection limit concentration of bromoacetic acid is 10.39 ng / mL, which is about 0.2078 ng; and the quantitative limit concentration is 20.77 ng / mL, which is about 0.4154 ng.

[0068] The present invention provides a method for detecting bromoacetic acid, a genotoxic impurity, comprising: A) reacting a compound, a solvent, a base and a derivatization agent to obtain a test solution; the compound is 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid; reacting bromoacetic acid, a solvent, a base and a derivatization agent to obtain a reference solution; B) performing high performance liquid chromatography on the test solution and the reference solution to obtain a test chromatogram and a reference chromatogram; C) calculating the content of bromoacetic acid by an external standard method according to the concentration of the reference, 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. The method provided by the present invention has good specificity, good durability, low detection limit, low quantitative limit, high separation, and relatively accurate results. The method provided by the present invention can accurately detect various genotoxic impurities in raw materials 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

[0069] Figure 1 The HPLC spectrum obtained under the first elution condition of the present invention;

[0070] Figure 2 This is the HPLC spectrum obtained under the second elution condition of the present invention;

[0071] Figure 3 It is the HPLC spectrum obtained after adjusting the sample preparation method of the present invention;

[0072] Figure 4 is the chromatogram of the specificity-blank solvent;

[0073] Figure 5 is the chromatogram of the specificity-blank derivatization solution (Sys-1);

[0074] Figure 6 is the chromatogram of the specificity-reference substance derivatization solution (Sys-2);

[0075] Figure 7 is the chromatogram of the specificity-test sample derivatized solution (Sys-3);

[0076] Figure 8 It is the chromatogram of the specificity-test sample spiked derivatization solution (Sys-4);

[0077] Fig. 9 is the chromatogram of the recovery-spiked 50% solution (A-spl L-1);

[0078] Fig.10 is the chromatogram of the recovery-spiked 100% solution (A-spl M-1);

[0079] Fig.11 is the chromatogram of the recovery-spiked 150% solution (A-spl H-1);

[0080] Fig.12 It is the chromatogram of the detection limit solution LOD;

[0081] Fig.13 The chromatogram is the LOQ of the quantification limit solution;

[0082] Fig.14 It is a linear relationship graph. DETAILED DESCRIPTION

[0083] The present invention provides a method for detecting bromoacetic acid, a genotoxic impurity, 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 are all considered to be included in the present invention. The methods and applications of the present invention have been described by 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.

[0084] The following is a detailed description of the method for detecting the genotoxic impurity bromoacetic acid provided by the present invention in conjunction with the examples.

[0085] In the following examples, the batch number of bromoacetic acid is 20180911, which is sourced from WuXi AppTec New Drug Development Co., Ltd.; the batch number of compound 1 is H21715-31-P1, which is sourced from WuXi AppTec New Drug Development Co., Ltd.

[0086] Example 1 Detection method of genotoxic impurity bromoacetic acid

[0087] The preparation process of compound 1 (see WO2021249468A1 for details) uses methyl bromoacetate, which may be hydrolyzed to derive into bromoacetic acid. In order to ensure the safety of the finished product, a highly sensitive method is developed to detect its residues.

[0088] The ultraviolet absorption of bromoacetic acid is weak, and it is difficult to directly measure it using HPLC or GC methods. Therefore, a derivatization method is used to measure bromoacetic acid derivatives with strong ultraviolet absorption, thereby determining the content of bromoacetic acid.

[0089] By examining different amounts of alkali, different amounts of derivatization reagents and reaction times, optimizing elution conditions, sample preparation methods, etc., and examining the separation degree between the derivatization reagent and adjacent impurities, the analytical method for bromoacetic acid detection was finally determined. The chromatographic conditions are shown in Table 1.

[0090] Table 1 Chromatographic conditions

[0091]

[0092] (I) Screening of derivatization reaction conditions

[0093] (1) Preparation of stock solution

[0094] 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.

[0095] Triethylamine solution: Measure 1 mL of triethylamine into a 10 mL volumetric flask, add acetonitrile to dilute to the scale, and shake well to obtain.

[0096] Formic acid solution: Measure 1 mL into a 10 mL volumetric flask, add acetonitrile to dilute to the mark, and shake well.

[0097] Bromoacetic acid 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 bromoacetic acid stock solution ① with a concentration of 1 mg / mL.

[0098] Bromoacetic acid 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 bromoacetic acid stock solution ② with a concentration of 1 μg / mL.

[0099] 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.

[0100] (2) Investigation of reaction time

[0101] Preparation of reference substance derivatization solution: 870 μL of acetonitrile, 100 μL of bromoacetic acid stock solution ②, 25 μL of 4-phenylthiophenol stock solution, and 5 μL of triethylamine were added to the same 2 mL injection vial, mixed well, allowed to stand at room temperature for a certain period of time, and then 8 μL of formic acid was added to terminate the reaction.

[0102] Preparation of the test sample spiked derivatization solution: 770 μL of acetonitrile, 100 μL of the test sample stock solution, 100 μL of bromoacetic acid stock solution ②, 25 μL of 4-phenylthiophenol stock solution, and 5 μL of triethylamine were added to the same 2 mL injection vial, mixed well, allowed to stand at room temperature for a certain period of time, and then 8 μL of formic acid was added to terminate the reaction.

[0103] The results are shown in Table 2, which shows the test results of different reaction times.

[0104] Table 2 Test results of different reaction times

[0105] Different reaction times STD target peak area STD+SPL target peak area 20min 15626 13767 30min 15508 14351 40min 15902 14978 60min 15927 15634

[0106] The test results show that after the reaction time of the reference substance derivatization solution was extended for 20 minutes, the target peak area did not change significantly. However, the peak area of ​​the test substance spiked derivatization solution gradually increased with the extension of reaction time. Considering that it may be related to other reaction conditions, mainly the amount of alkali, the reaction time was further adjusted after the alkali amount was determined to improve efficiency.

[0107] (3) Investigation of different alkali amounts

[0108] 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 bromoacetic acid stock solution ②, 25 μL of 4-phenylthiophenol stock solution, and different volumes of triethylamine solution into the same 2mL injection vial, mix well, let stand at room temperature for 60 minutes, add 90 μL of formic acid solution, mix well, and then inject for detection. 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.

[0109] The results are shown in Table 3, which shows the test results of different alkali amounts.

[0110] Table 3 Test results of different alkali amounts

[0111] Amount of alkali added (μL) Target peak area 40 14647 50 14528 60 14645 70 14506

[0112] The experimental results showed that adjusting the amount of triethylamine solution added had no significant effect on the area of ​​the derived target peak. In order to improve the reaction efficiency, the amount of base was set at 70 μL for subsequent condition optimization.

[0113] (4) Investigation of the amount of derivatization reagent used

[0114] Preparation of the spiked derivatization solution of the test sample with different derivatization reagent dosages: Take an appropriate amount of acetonitrile and 100 μL of the test sample stock solution into the same 2 mL injection vial, mix well, let stand at room temperature for 60 min, add 90 μL of formic acid solution, mix well, and then inject for detection. The amount of acetonitrile added was adjusted according to the amount of 4-phenylthiophenol stock solution added to keep the final derivatization solution volume consistent.

[0115] The results are shown in Table 4, which shows the test results of different derivatization reagent dosages.

[0116] Table 4 Test results of different actual derivatization dosages

[0117] Derivatization reagent volume (μl) Target peak area 25 14677 40 14910

[0118] 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.

[0119] (5) Further investigation of reaction time

[0120] Preparation of the test sample spiked derivatization solution: 705 μL of acetonitrile, 100 μL of the test sample stock solution, 100 μL of bromoacetic acid stock solution ②, 25 μL of 4-phenylthiophenol stock solution, and 70 μL of triethylamine solution were added to the same 2 mL injection vial, mixed well, allowed to stand at room temperature for a certain period of time, and then 90 μL of formic acid was added to terminate the reaction.

[0121] The results are shown in Table 5, which shows the test results of further adjusting the reaction time.

[0122] Table 5 Test results of further adjusting the reaction time

[0123] Different reaction time (min) STD+SPL target peak area 20 12746 30 14677 40 14263

[0124] The test results show that after the test sample was spiked with the derivatization solution and reacted for 30 minutes, the peak area did not change significantly with the extension of the reaction time, so the derivatization reaction time was set at 30 minutes.

[0125] The role of formic acid is to adjust the acidity and alkalinity of the final derivatization solution, improve the peak shape of the derivatized target peak, and prevent splitting without adding acid. Under the current situation where the amount of formic acid added has no effect on the stability of the target derivatization peak, no further adjustment is made to the amount of formic acid added.

[0126] (6) Optimization of chromatographic conditions

[0127] The results were as follows: Figure 1 As shown, Figure 1 The HPLC spectrum obtained under the first elution condition of the present invention is Figure 1 It can be seen that the method provided in this application has good specificity.

[0128] The results were as follows: Figure 2 As shown, Figure 2 This is the HPLC spectrum obtained under the second elution condition of the present invention.

[0129] Table 6

[0130] Time (min) 0 20 22 32 32.1 38 A(％) 60 60 5 5 60 60 B(％) 40 40 95 95 40 40 B(Curve) / 4 / / / /

[0131] Depend on Figure 2 It can be seen that the method provided in this application has good specificity, and the peak elution is no different from the chromatographic condition results shown in Table 1.

[0132] (7) Adjustment of sampling method

[0133] Considering that the 10 mg / mL test sample stock solution requires a large amount of test sample, the sample preparation method was adjusted. The test sample solution was directly prepared into 1 mg / mL and derivatized in a volumetric flask. All solutions were simultaneously amplified into volumetric flasks according to the optimized conditions for derivatization reaction, and specific solutions were prepared. The results are shown in Figure 3 , Figure 3 This is the HPLC spectrum obtained after the sample preparation method of the present invention is adjusted. Considering the reaction in the volumetric flask, acetonitrile is used to make the final volume, so the triethylamine-acetonitrile solution is directly converted into the volume of triethylamine added according to the amount of triethylamine; the formic acid-acetonitrile solution is directly converted into the volume of formic acid added according to the amount of formic acid. After peak area comparison, there is no significant difference in the target peak area derived from the same concentration of the two sample preparation methods, and the specificity is good. The blank derivative solution and the test sample derivative solution have no significant interference on the target peak. The peak area of ​​the reference sample derivative solution is close to that of the spiked test sample derivative solution, and the recovery rate is normal.

[0134] Example 2

[0135] 2.1 Preparation of stock solution

[0136] 4-Phenylthiophenol stock solution-1: Take 25.256 mg of 4-phenylthiophenol, accurately weigh it into a 25 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0137] 4-Phenylthiophenol stock solution-2: Take 10.190 mg of 4-phenylthiophenol, accurately weigh it into a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0138] Bromoacetic acid stock solution 1-1: accurately weigh 10.652 mg of bromoacetic acid, place in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well;

[0139] Bromoacetic acid stock solution 1-2: Accurately weigh 100 μL of bromoacetic acid stock solution 1-1, place in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well;

[0140] Bromoacetic acid stock solution 2-1: Accurately weigh 10.987 mg of bromoacetic acid, place in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well;

[0141] Bromoacetic acid stock solution 2-2: Accurately weigh 100 μL of bromoacetic acid stock solution 2-1, place in a 10 mL brown volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well;

[0142] Bromoacetic acid stock solution 3-1: accurately weigh 10.545 mg of bromoacetic acid, place in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well;

[0143] Bromoacetic acid stock solution 3-2: Accurately weigh 100 μL of bromoacetic acid stock solution 3-1, place in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well;

[0144] Bromoacetic acid stock solution 4-1: accurately weigh 10.517 mg of bromoacetic acid, place in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well;

[0145] Bromoacetic acid stock solution 4-2: Accurately weigh 100 μL of bromoacetic acid stock solution 4-1, place in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0146] 2.2 Preparation of reference and sample solutions

[0147] 2.2.1 Preparation of specific solution

[0148] Blank: acetonitrile;

[0149] Sys-1: Accurately pipette 9 mL of acetonitrile, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain Sys-1;

[0150] Sys-2: Accurately pipette 9 mL of acetonitrile, 100 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain;

[0151] Sys-3: Take 10.372 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve it; then accurately transfer 250 μL of 4-phenylthiophenol stock solution-1 and 70 μL of triethylamine to the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid and make up to volume with acetonitrile to obtain;

[0152] Sys-4: Take 10.486 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately transfer 100 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine to the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid and make up to volume with acetonitrile to obtain Sys-4.

[0153] 2.2.2 Preparation of stability solution

[0154] Blank: acetonitrile;

[0155] Sta-Std: Accurately pipette 9 mL of acetonitrile, 100 μL of bromoacetic acid stock solution 1-2, 1250 μL of 4-phenylthiophenol stock solution, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain Sta-Std.

[0156] Sta-spl: Take 10.486 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately pipette 100 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid and make up to volume with acetonitrile to obtain;

[0157] 2.2.3 Preparation of precision solution

[0158] Blank: acetonitrile;

[0159] Std-1: Accurately pipette 9 mL of acetonitrile, 100 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain;

[0160] Std-2: Accurately pipette 9 mL of acetonitrile, 100 μL of bromoacetic acid stock solution 2-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain;

[0161] 2.2.4 Accuracy and repeatability solution preparation

[0162] Blank: Acetonitrile

[0163] Std-1: Accurately pipette 9 mL of acetonitrile, 100 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain;

[0164] A-spl-1: Take 10.372 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve it; then accurately transfer 250 μL of 4-phenylthiophenol stock solution-1 and 70 μL of triethylamine to the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid and make up to volume with acetonitrile to obtain;

[0165] A-spl-2: Take 10.448 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve it; then accurately transfer 250 μL of 4-phenylthiophenol stock solution-1 and 70 μL of triethylamine to the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid and make up to volume with acetonitrile to obtain;

[0166] A-spl L-1: Take 10.485 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately pipette 50 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0167] A-spl L-2: Take 10.447 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately pipette 50 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0168] A-spl L-3: Take 10.563 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately pipette 50 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0169] A-spl M-1: Take 10.571 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve it; then accurately pipette 100 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0170] A-spl M-2: Take 10.202 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve it; then accurately pipette 100 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0171] A-spl M-3: Take 10.486 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately pipette 100 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0172] A-spl H-1: Take 10.408 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately pipette 150 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0173] A-spl H-2: Take 10.521 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately pipette 150 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0174] A-spl H-3: Take 10.671 mg of compound 1, accurately weigh it into a 10 mL brown volumetric flask, add 9 mL of acetonitrile to dissolve; then accurately pipette 150 μL of bromoacetic acid stock solution 1-2, 250 μL of 4-phenylthiophenol stock solution-1, and 70 μL of triethylamine into the same volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0175] 2.4.5 Preparation of System Suitability Solution 2

[0176] Blank: Acetonitrile

[0177] Std-1: Accurately pipette 9 mL of acetonitrile, 100 μL of bromoacetic acid stock solution 3-2, 250 μL of 4-phenylthiophenol stock solution-2, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain;

[0178] Std-1: Accurately pipette 9 mL of acetonitrile, 100 μL of bromoacetic acid stock solution 4-2, 250 μL of 4-phenylthiophenol stock solution-2, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain;

[0179] 2.4.6 Limit of Detection (LOD) and Limit of Quantitation (LOQ) Solution Preparation

[0180] Blank: acetonitrile;

[0181] LOD: Accurately pipette 9 mL of acetonitrile, 10 μL of bromoacetic acid stock solution 3-2, 250 μL of 4-phenylthiophenol stock solution-2, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0182] LOQ: Accurately pipette 9 mL of acetonitrile, 20 μL of bromoacetic acid stock solution 3-2, 250 μL of 4-phenylthiophenol stock solution-2, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and make up to volume with acetonitrile to obtain;

[0183] 2.2.7 Preparation of linear solution

[0184] Blank: Acetonitrile

[0185] Lin-20%: Accurately pipette 9 mL of acetonitrile, 20 μL of bromoacetic acid stock solution 3-2, 250 μL of 4-phenylthiophenol stock solution-2, and 70 μL of triethylamine into the same 10 mL brown volumetric flask, vortex for about 30 seconds, react at room temperature for 30 minutes, add 90 μL of formic acid, and adjust the volume with acetonitrile to obtain;

[0186] Lin-50%: Accurately pipette 9mL of acetonitrile, 50μL of bromoacetic acid stock solution 3-2, 250μL of 4-phenylthiophenol stock solution-2, and 70μL of triethylamine into the same 10mL brown volumetric flask, vortex for about 30s, react at room temperature for 30min, add 90μL of formic acid and make up to volume with acetonitrile to obtain;

[0187] Lin-100%: Accurately pipette 9mL of acetonitrile, 100μL of bromoacetic acid stock solution 3-2, 250μL of 4-phenylthiophenol stock solution-2, and 70μL of triethylamine into the same 10mL brown volumetric flask, vortex for about 30s, react at room temperature for 30min, add 90μL of formic acid, and make up to volume with acetonitrile to obtain;

[0188] Lin-150%: Accurately pipette 9mL of acetonitrile, 150μL of bromoacetic acid stock solution 3-2, 250μL of 4-phenylthiophenol stock solution-2, and 70μL of triethylamine into the same 10mL brown volumetric flask, vortex for about 30s, react at room temperature for 30min, add 90μL of formic acid, and make up to volume with acetonitrile to obtain;

[0189] Lin-200%: Accurately pipette 9mL of acetonitrile, 200μL of bromoacetic acid stock solution 3-2, 250μL of 4-phenylthiophenol stock solution-2, and 70μL of triethylamine into the same 10mL brown volumetric flask. Vortex for about 30s and react at room temperature for 30min. Add 90μL of formic acid and make up to volume with acetonitrile to obtain Lin-200%.

[0190] 2.3 Use the chromatographic conditions in Table 1 to verify the methodology.

[0191] 2.3.1 Exclusivity

[0192] 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. See Figures 4 to 8 Records of Figure 4 is the chromatogram of the specificity-blank solvent; Figure 5 is the chromatogram of the specificity-blank derivatization solution (Sys-1); Figure 6 is the chromatogram of the specificity-reference substance derivatization solution (Sys-2); Figure 7 is the chromatogram of the specificity-test sample derivatized solution (Sys-3);

[0193] Figure 8 The chromatogram is of the specificity-test sample spiked derivatization solution (Sys-4).

[0194] Table 6-1

[0195]

[0196] Conclusion: There is no interference between the blank derivatization solution and the test sample derivatization solution at the target chromatographic peak, and the minimum separation between the target chromatographic peak and nearby impurity peaks is 1.7, indicating that the method has good specificity.

[0197] 2.3.2 Injection precision

[0198] 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.

[0199] Table 6-2

[0200]

[0201] Conclusion: Std-1 solution was injected 6 times continuously, the RSD of target peak area was 0.8%, and the injection precision was good.

[0202] 2.3.3 Solution stability

[0203] The stability solution stored at room temperature was sampled and the changes in the content of bromoacetic acid in the reference solution and the spiked test solution within 19 hours compared with 0 hours were examined. The system suitability results are shown in Table 6-3, and the stability results are shown in Table 6-4, where Std0h refers to the first injection data of "Table 6-3 System Suitability Results", and Std3.0h refers to the sixth injection data of "Table 6-3 System Suitability Results".

[0204] Table 6-3

[0205]

[0206] Table 6-4

[0207]

[0208] Conclusion: The system suitability results meet the requirements; the bromoacetic acid content of the reference solution and the test solution at 19 hours is between 80.0% and 120.0% compared with that at 0 hours, and the solution is stable.

[0209] 2.3.4 Accuracy and repeatability

[0210] Take the accuracy and repeatability solutions, 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 6-3, the test results of the unspiked test solution are shown in Table 6-5, and the accuracy and repeatability results are shown in Table 6-6, where A-spl M-3 refers to Sta-spl9.5 hour data. See the results Fig. 9 , Fig.10 and Fig.11 , Fig. 9 is the chromatogram of the recovery-spiked 50% solution (A-spl L-1); Fig.10 is the chromatogram of the recovery-spiked 100% solution (A-spl M-1); Fig.11 The chromatogram is the recovery-spiked 150% solution (A-spl H-1).

[0211] Table 6-5 Test results of unspiked test solution

[0212]

[0213] Table 6-6 Accuracy and repeatability test results

[0214]

[0215] Conclusion: The system suitability results meet the requirements. The recoveries of bromoacetic acid at three concentration levels are all in the range of 97.7% to 102.0%. The RSD of the recoveries of nine solutions is 1.5%, which meets the requirements.

[0216] 2.3.5 Limit of detection (LOD) and limit of quantification (LOQ)

[0217] Take the detection limit solution and inject it into 3 needles, take the quantitative 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 6-7, and the detection limit solution test results are shown in Table 6-8 and Fig.12 The results of the quantitative limit solution are shown in Tables 6-9 and Fig.13 , Fig.12 It is the chromatogram of the detection limit solution LOD; Fig.13 The chromatogram is the LOQ of the quantification limit solution.

[0218] Table 6-7 System suitability results

[0219]

[0220] Table 6-8 Detection limit experimental results

[0221]

[0222] Table 6-9 Quantitation limit experimental results

[0223]

[0224] Conclusion: The detection limit concentration is 10.39 ng / mL (equivalent to 10% of the limit concentration), and the detection limit is 0.2078 ng; the quantification limit concentration is 20.77 ng / mL (equivalent to 20% of the limit concentration), and the quantification limit is 0.4154 ng. The signal-to-noise ratio and peak area RSD both meet the requirements.

[0225] 2.3.6 Linearity and Range

[0226] Take each linear solution and inject 3 needles continuously, record the chromatogram and peak area, among which Lin-20% refers to the data of the three needles after LOQ, the system suitability results are the same as Table 6-7, the linear solution determination results are shown in Table 6-10, and the linear relationship diagram is shown in Fig.14 , Fig.14 It is a linear relationship graph.

[0227] Table 6-10

[0228]

[0229] Conclusion: The concentration of bromoacetic acid derivatives has a good linear relationship with the peak area in the range of 20.77 ng / mL to 207.74 ng / mL. 2 It is 1.000, which meets the requirements.

[0230] The summary of the methodological validation is shown in Table 7:

[0231] Table 7 Summary of method validation

[0232]

[0233] Example 3 Sample determination

[0234] 1. Reagents and reagents: acetonitrile, triethylamine, formic acid, 4-phenylthiophenol, ultrapure water.

[0235] 2. Instruments and appliances: Liquid chromatograph equipped with DAD or VWD detector, ultrapure water instrument, Luna5u Phenyl-Hexyl (4.6×250mm, 5μm), electronic analytical balance, vortex machine, pipette, and standard volumetric flask.

[0236] 3. Preparation of stock solution: 4-phenylthiophenol stock solution: Take about 25 mg of 4-phenylthiophenol, accurately weigh and place in a 25 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well to obtain;

[0237] Reference substance stock solution: take about 10 mg of bromoacetic acid, 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, shake well.

[0238] 4. Test solution: Weigh about 10 mg of compound 1 sample, weigh accurately, place in a 10 ml brown volumetric flask, accurately pipette 9.0 ml of acetonitrile to dissolve, then accurately pipette 70 μl of triethylamine and 250 μl of 4-phenylthiophenol stock solution into the same volumetric flask, vortex for about 30 seconds, react at room temperature for about 30 minutes, add 90 μl of formic acid, make up to volume with acetonitrile, and mix well. Prepare blank solution in the same way.

[0239] 5. Reference substance solution: Accurately pipette 9.0 ml of acetonitrile, 70 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. Vortex for about 30 seconds, react at room temperature for about 30 minutes, add 90 μl of formic acid, make up to volume with acetonitrile, and mix well.

[0240] 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 9.0 ml of acetonitrile to dissolve it, then accurately pipette 20 μl of the reference substance stock solution, 70 μl of triethylamine, and 250 μl of 4-phenylthiophenol stock solution into the same volumetric flask, vortex for about 30 seconds, react at room temperature for about 30 minutes, add 90 μl of formic acid, make up to volume with acetonitrile, and mix well.

[0241] 7. Operation method: Detect according to the general rules of the fourth part of the Chinese Pharmacopoeia 2020 edition 0512. Use Luna 5u Phenyl-Hexyl (4.6×250mm, 5μm) 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 linear gradient elution is performed according to Table 8; the detection wavelength is 278nm; the column temperature is 40℃.

[0242] Table 8 Linear gradient elution conditions

[0243]

[0244]

[0245] 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 bromoacetic acid 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 bromoacetic acid derivative peak in the test solution, calculate the content of bromoacetic acid by peak area according to the external standard method.

[0246] 8. Calculation

[0247] Bromoacetic acid content

[0248]

[0249] in:

[0250] A S = the peak area of ​​bromoacetic acid derivatives in the sample solution spectrum;

[0251] A R = the average peak area of ​​the bromoacetic acid derivative peak in the reference solution;

[0252] C S = sample concentration (mg / ml);

[0253] C R = concentration of bromoacetic acid in the reference solution (ng / ml);

[0254] 9. Limit: The content of bromoacetic acid shall not exceed 50ppm.

[0255] The results are shown in Table 9, which shows the results of bromoacetic acid detection in different batches of samples.

[0256] Table 9 Analytical methods for detection of bromoacetic acid in different batches of samples

[0257] Impurity Name limit Batch 1 Batch 2 Bromoacetic acid 50ppm Not detected <20ppm

[0258] Conclusion: All validation results were within the acceptable range, indicating that the HPLC method was suitable for the detection of bromoacetic acid in bulk drug compound 1 samples.

[0259] The above are only preferred embodiments 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 principles 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 bromoacetic acid, a genotoxic impurity in 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, a base and a derivatizing agent to obtain a test solution; the compound is 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1,2,4-triazol-3-yl)-thio)acetic acid; reacting bromoacetic acid, a solvent, a base and a derivatization agent to obtain a reference substance solution; B) 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 chromatographic parameters are as follows: Mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile; The elution procedure is: 0-22min, phase A 60%, phase B 40%; 22-23min: Phase A 60%-5%, Phase B 40%-95%; 23-30min: Phase A 5%, Phase B 95%; 30~30.1min Phase A 5%~60%, Phase B 95%~40%; 30.1~38min Phase A 60%, Phase B 40%; Alternatively, the elution program is: 0-20min, phase A 60%, phase B 40%; 20-22min: Phase A 60%-5%, Phase B 40%-95%; 22-32min: Phase A 5%, Phase B 95%; 32~32.1min Phase A 5%~60%, Phase B 95%~40%; 32.1~38min Phase A 60%, Phase B 40%; C) calculating the content of bromoacetic acid 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 in the chromatogram.

2. The detection method according to claim 1, characterized in that: The derivatization agent is selected from 4-phenylthiophenol; The base is selected from triethylamine.

3. The detection method according to claim 1, characterized in that: The reaction temperature in step A) is 15-35°C; the reaction time is 20-40 minutes.

4. The detection method according to claim 1, characterized in that: Step A) further comprises, after the compound, solvent, base and derivatization agent react, adjusting the pH value with formic acid; and After the reaction of bromoacetic acid, solvent, base and derivatization agent, formic acid is used to adjust the pH value.

5. The detection method according to claim 1, characterized in that: The chromatographic column is Luna 5uPhenyl-Hexyl, the chromatographic column specification is 4.6×250mm, 5μm; the chromatographic column temperature is 40°C.

6. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8-1.2 mL / min; the injection volume is 10-100 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 bromoacetic acid is in a good linear relationship between 20.77 ng / ml and 207.74 ng / ml.

9. The detection method according to claim 1, characterized in that: The detection limit concentration of the bromoacetic acid is 10.39 ng / mL; the quantification limit concentration is 20.77 ng / mL.

10. The detection method according to claim 1, characterized in that: The solvent in step A) is acetonitrile.

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