Method for detecting compound-SMA and compound-A

Abstract

The invention provides a method for detecting a compound-SMA and a compound-A in a compound 2-((5-bromo-4-(2-chloro-4-cyclopropylnaphthyl-1-yl)-4H-1, 2, 4-triazole-3-yl)-sulfur) acetic acid, which comprises the following steps: dissolving the compounds in a solvent to obtain a test solution; the compound is-((5-bromo-4-(2-chloro-4-cyclopropyl naphthyl-1-yl)-4H-1, 2, 4-triazole-3-yl)-sulfur) acetic acid, and the structural formula of the compound is shown in the description. Dissolving the compound-SMA and the compound-A with a solvent to obtain a reference solution; and respectively carrying out high performance liquid chromatography-mass spectrometry 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 202311100804.4 and invention name “Detection Method of Compound-SMA and Compound-A”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of detection technology, and in particular to a detection method for compound-SMA and compound-A. Background Art

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

[0004]

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

[0006] Therefore, it is very necessary to develop a detection method for compound-SMA and compound-A to accurately reflect the content of compound-SMA and compound-A in the raw material or preparation of compound 1. The structures of the two are as follows

[0007] Summary of the invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a detection method for compound-SMA and compound-A. The detection method provided by the present invention has good separation, specificity and durability, high responsiveness and accurate results.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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 detection method for compound 1, compound-SMA and compound-A. 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.

[0014] The present invention provides a method for detecting compound-SMA and compound-A 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:

[0015] A) dissolving a compound in a solvent 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;

[0016] B) dissolving compound-SMA and compound-A in a solvent to obtain a reference solution;

[0017] C) subjecting the test solution and the reference solution to high performance liquid chromatography-mass spectrometry to obtain a test sample chromatogram and a reference sample chromatogram;

[0018] The specific chromatographic parameters are as follows:

[0019] Mobile phase A: 0.1% formic acid solution, mobile phase B: 0.1% formic acid acetonitrile solution;

[0020] The elution procedure is:

[0021] 0-10min, phase B 50-80%;

[0022] 10-16min Phase B 80%;

[0023] 16~16.1min Phase B 80~50%;

[0024] 16.1~24min Phase B 50%;

[0025] D) calculating the contents of compound-SMA and compound-A 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 in the test solution corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step C).

[0026] The present invention provides a method for detecting compound-SMA and compound-A 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 firstly dissolving the compound in a solvent to obtain a test solution.

[0027] 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.

[0028] The structure is shown in formula (I):

[0029]

[0030] The solvent of the present invention includes but is not limited to acetonitrile.

[0031] Compound-SMA and compound-A are dissolved in a solvent to obtain a reference solution.

[0032] The solvent of the present invention includes but is not limited to acetonitrile.

[0033] The experimental results showed that the concentration of the compound-SMA was in a good linear relationship between 3.31ng / ml and 33.12ng / ml;

[0034] The concentration of the compound-A showed a good linear relationship in the range of 3.25 ng / ml-32.46 ng / ml.

[0035] The present invention also includes the preparation of the sensitivity solution, which is specifically: adding the reference substance to dimethyl sulfoxide to dissolve it, and then diluting it with a solvent. The solvent includes but is not limited to acetonitrile.

[0036] The test solution and the reference solution are respectively subjected to high performance liquid chromatography-mass spectrometry to obtain a test sample chromatogram and a reference sample chromatogram; 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), the contents of compound-SMA and compound-A are calculated by the external standard method.

[0037] The present invention does not limit the above external standard method for calculating the contents of compound-SMA and compound-A, which is well known to those skilled in the art.

[0038] The specific chromatographic parameters of the present invention are as follows:

[0039] Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile;

[0040] The elution procedure is:

[0041] 0-10min, phase B 50-80%;

[0042] 10-16min Phase B 80%;

[0043] 16~16.1min Phase B 80~50%;

[0044] 16.1~24min Phase B 50%;

[0045] The present invention achieves good baseline separation, good separation of each peak and stable baseline by selecting the mobile phase gradient.

[0046] In the present invention, the chromatographic column is C18, and the chromatographic column specifications are 4.6×150 mm, 5 μm; more preferably, the chromatographic column is Waters C18.

[0047] The temperature of the chromatographic column of the present invention is preferably 40°C.

[0048] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above 40°C condition.

[0049] In the present invention, the flow rate of the mobile phase is 0.5 mL / min, the injection volume is 5 μL, and the injection plate temperature is 5°C.

[0050] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 0.5 ml / min, which was the most preferred solution.

[0051] In some preferred embodiments of the present invention, the mass spectrometry conditions are:

[0052] Ion source: ESI; Scan mode: POSITIVE, SIM; Dwell Time 200ms, Frag: 135volts.

[0053] In some preferred embodiments of the present invention, the mass spectrometry conditions are:

[0054] GAS 350℃, gas flow rate: 12.0L / min, Nebulizer 60psi; Capillary Voltage 4000volts.

[0055] In some preferred embodiments of the present invention, the characteristic ion peaks are specifically: 184.1 for compound-SMA and 218.1 for compound-A.

[0056] In some preferred embodiments of the present invention, the valve method is specifically:

[0057] 0~6.5to Waste; 6.5~8.5to Ms; 8.5~13.5to Waste; 13.5~15.5to Ms; 15.5~24to Waste.

[0058] In the present invention, the detection limit concentration of the compound-SMA and compound-A is 1.66 ng / ml; the quantification limit concentration is 3.31 ng / ml;

[0059] In the present invention, the detection limit concentration of compound-A is 1.62 ng / ml; the quantification limit concentration is 3.25 ng / ml.

[0060] The invention has low detection limit, low quantitative limit, high separation degree and good effect.

[0061] The present invention provides a method for detecting compound SMA and compound A 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) dissolving the compound in a solvent 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; B) dissolving the compound in a solvent 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; Compound-SMA and compound-A are dissolved in a solvent to obtain a reference solution; C) the test solution and the reference solution are respectively subjected to high performance liquid chromatography-mass spectrometry detection 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), the content of compound-SMA and compound-A is calculated by an 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

[0062] Figure 1 It is a specificity-blank solution, the upper figure is a chromatogram, and the lower figure is a mass spectrum;

[0063] Figure 2 It is a specificity-reference solution, the upper figure is a chromatogram, and the lower figure is a mass spectrum;

[0064] Figure 3 For specificity-test solution, the upper figure is the chromatogram, and the lower figure is the mass spectrum;

[0065] Figure 4 The specificity-test sample spiked solution, the upper figure is the chromatogram, and the lower figure is the mass spectrum;

[0066] Figure 5 It is the specificity-compound-SMA reference solution, the upper figure is the chromatogram, and the lower figure is the mass spectrum;

[0067] Figure 6 It is the specificity-compound-A reference solution, the upper figure is the chromatogram, and the lower figure is the mass spectrum;

[0068] Figure 7 The upper figure is the chromatogram and the lower figure is the mass spectrum;

[0069] Figure 8 The figure above is the chromatogram and the figure below is the mass spectrum.

[0070] Fig. 9 is a linear relationship graph;

[0071] Fig.10 is the recovery rate - spiked 50% solution, the upper figure is the chromatogram, and the lower figure is the mass spectrum;

[0072] Fig.11 is the recovery rate - spiked 100% solution, the upper figure is the chromatogram, and the lower figure is the mass spectrum;

[0073] Fig.12 The upper figure is the chromatogram and the lower figure is the mass spectrum. DETAILED DESCRIPTION

[0074] The present invention provides a method for detecting compound-SMA and compound-A. 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.

[0075] In order to further illustrate the present invention, the detection method of compound-SMA and compound-A provided by the present invention is described in detail below in conjunction with examples.

[0076] Compound-SMA batch number is 20170715 and the source is Shanghai Feibai Chemical Technology Co., Ltd.;

[0077] Compound-A batch number is 20181001 and the source is Jinan Sanzhi Pharmaceutical Technology Co., Ltd.;

[0078] Compound 1, batch number H21715-31-P1, was obtained from WuXi AppTec.

[0079] Example 1 Detection Method of Genotoxic-SMA and Compound-A Impurities

[0080] Compound-SMA and Compound-A are both genotoxic impurities with high risks in the preparation process of Compound 1 (see WO2021249468A1 for details). In order to ensure the safety of the finished product, an LCMS method is developed in the final product for control.

[0081] 1. Reagents and reagents: acetonitrile, formic acid, dimethyl sulfoxide (DMSO), ultrapure water.

[0082] 2. Instruments and equipment: Liquid phase mass spectrometer, chromatographic column Waters C18 (4.6×150mm, 5μm), electronic analytical balance, pipette, standard volumetric flask.

[0083] 3. Test solution: Take about 5 mg of compound 1 sample, weigh accurately, place in a 10 ml volumetric flask, dissolve with solvent (acetonitrile) and quantitatively dilute to the scale, shake well.

[0084] 4. Reference solution:

[0085] Take about 5 mg of compound-SMA and compound-A, weigh accurately, put them in a 10 ml volumetric flask, transfer 1 ml of DMSO to dissolve it, add solvent to dilute to the scale, shake well, and mark it as stock solution 1; accurately transfer 33 μl of stock solution 1 to a 10 ml volumetric flask, add solvent to the scale, shake well, and mark it as stock solution 2; accurately transfer 100 μl of stock solution 2 to a 10 ml volumetric flask, add solvent to the scale, shake well, and mark it as reference solution.

[0086] 5. Sensitivity solution: Accurately pipette 20 μl of stock solution 2 into a 10 ml volumetric flask, add solvent to the mark, shake well, and mark as sensitivity solution.

[0087] 6. Operation method: According to the mass spectrometry method 0431 of the fourth volume of the Chinese Pharmacopoeia 2020 edition. C18 (4.6×150mm, 5μm) was used as the chromatographic column; 0.1% formic acid solution was used as the mobile phase A, 0.1% formic acid acetonitrile solution was used as the mobile phase B, the flow rate was 0.5 ml per minute, and linear gradient elution was performed according to the table below; the column temperature was 40°C, and the injection plate temperature was 5°C; the mass spectrometry conditions were: ion source ESI, scanning mode POSITIVE, SIM, Dwell Time 200ms, Frag: 135volts, GAS 350°C, gas flow rate: 12.0L / min, characteristic ion peaks: compound-SMA184.1, compound-A218.1.

[0088] The mobile phase gradient is:

[0089]

[0090] Take 5μl of blank solvent and inject it into the liquid mass spectrometer. There should be no interference. Take another 5μl of sensitivity solution and inject it into the liquid mass spectrometer. Record the chromatogram. The signal-to-noise ratio of compound-SMA and compound-A peaks should be no less than 10. Then accurately measure 5μl of reference solution and test solution respectively, inject them into the liquid mass spectrometer respectively, and record the chromatogram. If compound-SMA and compound-A peaks appear in the test solution, calculate the content of compound-SMA and compound-A by peak area according to the external standard method.

[0091] 7. Calculation

[0092] Content of Compound-SMA and Compound-A

[0093]

[0094] in:

[0095] A S = Compound-SMA or Compound-A peak area in the sample solution spectrum

[0096] A R = Average peak area of ​​Compound-SMA or Compound-A peak in reference solution

[0097] C S = Sample concentration (mg / ml)

[0098] C R = Concentration of Compound-SMA or Compound-A in the reference solution (ng / ml)

[0099] 8. Limit: The content of compound-SMA and compound-A shall not exceed 33ppm.

[0100] Table 3. Analytical methods for detection of compound-SMA or compound-A in different batches of samples

[0101] Impurity Name limit Batch 1 Batch 2 Compound-SMA Should be ≤33ppm Not detected Not detected Compound-A Should be ≤33ppm 16.0ppm 15.7ppm

[0102] Conclusion: The specificity, detection limit and quantification limit, linearity and range, accuracy and solution stability of the analytical method were verified by LCMS method, and all the verification results were within the acceptable range, indicating that the LCMS method is suitable for the detection of compound-SMA and compound-A in bulk drug samples.

[0103] Example 2

[0104] 2.8 Use the chromatographic conditions in Table 1 to verify the methodology.

[0105] 2.8.1 Exclusivity

[0106] Take the specific solution, inject it separately, and record the chromatogram. Record the retention time of compound-SMA and compound-A, and use the retention time to locate the impurity. The results are shown in Table 6.

[0107]

[0108] Among them, sys-1 is the dilution solution, the std compound-SMA concentration is 16.4865ng / ml; the std compound-A concentration is 16.2992ng / ml; the compound-SMA concentration in sys-2 is 16.6835ng / ml, and the compound-A concentration is 16.2414ng / ml;

[0109] Sys-3: Take 5.074 mg of compound 1 into a 10 mL volumetric flask, add diluent to the mark and shake well.

[0110] Sys-4 is to take 5.067 mg of compound 1 into a 10 mL volumetric flask, add diluent to dissolve, accurately transfer 100 μL of stock-1 into the same volumetric flask, add diluent to the mark, and shake well.

[0111] Figure 1 It is the specificity-blank solution; Figure 2 It is a specificity-control solution; Figure 3 It is a specific test solution; Figure 4 It is the specificity-test sample spike solution; Figure 5 It is the specificity-compound-SMA reference solution; Figure 6 It is the specificity-Compound-A reference solution.

[0112] Conclusion: Neither the blank solution nor the sample solution had obvious interference with the target peak, and the method had good specificity.

[0113] 2.8.2 Injection precision

[0114] Take the precision solution and inject 6 times continuously, and record the chromatogram. The results of the precision solution injection are shown in Table 6.

[0115] The precision solution is as follows: the concentration of compound-SMA is 16.5620 ng / ml; the concentration of compound-A is 16.2285 ng / ml.

[0116] Table 6 Injection precision results

[0117]

[0118] Conclusion: After 6 consecutive injections of the solution, the RSD of the target peak area of ​​compound-SMA was 1.4%, and the RSD of the target peak area of ​​compound-A was 1.5%, and the injection precision was good.

[0119] 2.8.3 Solution stability

[0120] The stability solution stored at 5°C was sampled and the changes in the contents of compound-SMA and compound-A in the reference solution and the spiked test solution compared with 0 hour were examined within 16 hours. The system suitability results are shown in Table 7, and the stability results are shown in Table 8-1 and Table 8-2.

[0121] The stability solution is configured with a compound-SMA concentration of 16.6835 ng / ml and a compound-A concentration of 16.2414 ng / ml.

[0122] Table 7 System suitability results

[0123]

[0124] Table 8-1 Compound 1--SMA stability test results

[0125]

[0126] Table 8-2 Compound 1--A stability test results

[0127]

[0128] Conclusion: The system suitability results meet the requirements; the impurity contents of the reference solutions of compound-SMA and compound-A and the spiked solutions of the test samples at 16 hours were all between 80.0% and 120.0% compared with those at 0 hours, and the solutions were stable.

[0129] 2.8.4 Accuracy

[0130] Stock-1: Weigh 5.044 mg of compound-SMA and 5.049 mg of compound-A respectively into the same 10 ml volumetric flask, add 1 ml of DMSO to dissolve, then add diluent to dilute to the scale, shake well, then accurately measure 33 μL into a 10 ml volumetric flask, add diluent to dilute to the scale, and shake well.

[0131] Preparation of unspiked test solution: Take about 5 mg of compound 1, weigh accurately, place in a 10 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well. Prepare 2 copies in parallel.

[0132] Sample addition - low concentration: Take about 5 mg of compound 1, weigh accurately, and place in a 10 ml volumetric flask. Accurately pipette 50 μL of Stock-1 into the same volumetric flask, add diluent to dissolve and dilute to the mark, shake well. Prepare 3 copies in parallel.

[0133] Sample addition - medium concentration: Take about 5 mg of compound 1, weigh accurately, and place in a 10 ml volumetric flask. Accurately pipette 100 μL of Stock-1 into the same volumetric flask, add diluent to dissolve and dilute to the mark, shake well. Prepare 3 copies in parallel.

[0134] Sample addition - high concentration: Take about 5 mg of compound 1, weigh accurately, and place in a 10 ml volumetric flask. Accurately pipette 150 μL of Stock-1 into the same volumetric flask, add diluent to dissolve and dilute to the mark, shake well. Prepare 3 copies in parallel.

[0135] Take the accuracy solution 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 of compound-SMA are shown in Table 9-1, the test results of the unspiked test solution of compound-A are shown in Table 9-2, and the accuracy and repeatability results are shown in Tables 10-1 and 10-2.

[0136] Fig.10 is the recovery rate - spiked with 50% solution; Fig.11 is the recovery rate - spiked 100% solution; Fig.12 The recovery is the 150% spike solution.

[0137] Table 9-1 Test results of compound-SMA unspiked test solution

[0138]

[0139] Table 9-2 Test results of unspiked test solution of compound A

[0140]

[0141] Table 10-1 Compound-SMA accuracy test results

[0142]

[0143] Table 10-2 Compound-A accuracy test results

[0144]

[0145] Conclusion: The system suitability results meet the requirements; the recoveries of compound-SMA content at three concentration levels were all in the range of 81.2% to 111.3%, and the RSD of the recoveries of 9 solutions was 12.4%, which all met the requirements; the recoveries of compound-A content at three concentration levels were all in the range of 104.1% to 106.2%, and the RSD of the recoveries of 9 solutions was 0.7%, which all met the requirements.

[0146] 2.8.5 Limit of detection (LOD) and limit of quantification (LOQ)

[0147] Preparation of detection limit solution: Accurately pipette 10 μL of Stock-1 into a 10 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0148] Preparation of quantitative limit solution: Accurately pipette 20 μL of Stock-1 into a 10 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0149] Figure 7 is the detection limit spectrum; Figure 8 The quantitation limit spectrum.

[0150] The detection limit solution was injected into 3 needles, and the quantitation limit solution was injected into 6 needles, and the chromatogram, peak area and signal-to-noise ratio were recorded. The system suitability results are shown in Table 7, the detection limit solution determination results are shown in Table 11, and the quantitation limit solution determination results are shown in Table 12.

[0151] Table 11 Test line experimental results

[0152]

[0153] Table 12 Quantitation limit experimental results

[0154]

[0155] Conclusion: The system suitability results meet the composite requirements; the detection limit concentration of compound-SMA is 1.66ng / ml; the quantitative limit concentration is 3.31ng / ml, and the signal-to-noise ratio and peak area RSD meet the requirements;

[0156] The detection limit concentration of compound-A is 1.62ng / ml; the quantification limit concentration is 3.25ng / ml, and the signal-to-noise ratio and peak area RSD both meet the requirements;.

[0157] 2.8.6 Limitation and scope

[0158] Lin-LOQ: This is the quantitative limit solution mentioned above.

[0159] Lin-50%: Accurately pipette 50μL of Stock-1 into a 10ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0160] Lin-100%: Accurately pipette 100μL of Stock-1 into a 10ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0161] Lin-150%: Accurately pipette 150μL of Stock-1 into a 10ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0162] Lin-5 200%: Accurately pipette 200μL of Stock-1 into a 10ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0163] Take each linear solution and inject 3 needles, record the chromatogram and peak area. Lin-LOQ refers to the data of the three needles after LOQ, the system suitability results are the same as Table 7, the compound-SMA linear solution determination results are shown in Table 13, and the linear relationship diagram is shown in Fig. 9 , Fig. 9 is a linear relationship diagram between compound-SMA and compound-A; the results of the linear solution determination of compound-A are shown in Table 14,

[0164] Table 13 Compound-SMA linearity and range test results

[0165]

[0166]

[0167] Table 14 Compound-A linearity and range test results

[0168]

[0169] Conclusion: The concentration of compound-SMA has a good linear relationship with the peak area in the range of 3.31ng / ml to 33.12ng / ml. 2 The peak area of ​​compound A has a good linear relationship in the range of 3.25 ng / ml to 32.46 ng / ml. 2 It is 0.9996, which meets the requirements.

[0170] 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 compound-SMA and compound-A in 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) dissolving a compound in a solvent 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) dissolving compound-SMA and compound-A in a solvent to obtain a reference solution; C) subjecting the test solution and the reference solution to high performance liquid chromatography-mass spectrometry to obtain a test sample chromatogram and a reference sample chromatogram; The specific chromatographic parameters are as follows: Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; The elution procedure is: 0-10min, phase B 50-80%; 10-16min Phase B 80%; 16~16.1min Phase B 80~50%; 16.1~24min Phase B 50%; D) calculating the contents of compound-SMA and compound-A 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 in the test solution corresponding to the reference substance 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 mass spectrometry conditions are: Ion source: ESI; Scan mode: POSITIVE, SIM; DwellTime 200ms, Frag: 135volts.

3. The detection method according to claim 1, characterized in that: The mass spectrometry conditions are: GAS 350℃, gas flow rate: 12.0L / min, Nebulizer 60psi; Capillary Voltage 4000volts.

4. The detection method according to claim 1, characterized in that: The characteristic ion peaks are: 184.1 for compound-SMA and 218.1 for compound-A.

5. The detection method according to claim 1, characterized in that: The chromatographic column is C18, and the specifications of the chromatographic column are 4.6×150 mm, 5 μm; the temperature of the chromatographic column is 40° C.

6. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase was 0.5 mL / min; the injection volume was 5 μL; and the injection plate temperature was 5° C.

7. The detection method according to claim 1, characterized in that: The concentration of -SMA showed a good linear relationship between 3.31ng / ml and 33.12ng / ml; The concentration of the compound-A showed a good linear relationship in the range of 3.25 ng / ml-32.46 ng / ml.

8. The detection method according to claim 1, characterized in that: The detection limit concentration of the compound-SMA and compound-A is 1.66 ng / ml; the quantification limit concentration is 3.31 ng / ml; The detection limit concentration of the compound-A is 1.62 ng / ml; the quantification limit concentration is 3.25 ng / ml.

9. The detection method according to claim 1, characterized in that: The valve method is specifically: 0~6.5to Waste; 6.5~8.5to Ms; 8.5~13.5to Waste; 13.5~15.5to Ms; 15.5~24toWaste.

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.

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