A method for detecting related substances in 2-aminothiophenol

Abstract

The present invention belongs to the field of pharmaceutical analysis technology, and specifically relates to a method for detecting related substances in 2-aminothiophenol. The present invention provides a method for detecting related substances in 2-aminothiophenol, and provides a reverse-phase HPLC assay method that can detect related substances in 2-aminothiophenol. The method has good specificity, linearity, precision, sensitivity, accuracy, and durability, and is suitable for quality control of 2-aminothiophenol.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a high performance liquid chromatography method for related substances in 2-aminothiophenol. Background Art

[0002] 2-Aminothiophenol is an important organic synthesis intermediate with a wide range of applications in various fields. It can serve as a key fragment in the construction of complex drug molecules. Through chemical reactions, it combines with other functional groups to form compounds with specific pharmacological activities. 2-Aminothiophenol, CAS number: 137-07-5, has the following structural formula:

[0003] .

[0004] Dotinurad is a drug used to treat gout and hyperuricemia. 2-Aminothiophenol is a key intermediate in the synthesis of dotinurad. Diltiazem is a non-dihydropyridine calcium channel blocker primarily used to treat hypertension, angina pectoris, and certain arrhythmias. 2-Aminothiophenol is also an important intermediate in the synthesis of diltiazem. 2-Aminothiophenol can also be used to synthesize other benzothiazole compounds, which have a wide range of biological activities in the pharmaceutical field and can be used to treat various diseases, including cancer and cardiovascular disease.

[0005] The synthetic process of 2-aminothiophenol is as follows:

[0006] .

[0007] 2-Aminothiophenol is easily oxidized and deteriorates in air, and there are few methods for detecting it. Zhang Weimin, "Determination of o-aminothiophenol," Liaoning Chemical Industry, 1994, pp. 61-62, discloses a method for determining o-aminothiophenol. The main methods for determining o-aminothiophenol include spectrophotometry, volumetry, and chromatography. Spectrophotometry is simple to operate but has a large relative error, while volumetry has a relative error of 0.25%. Chromatography is primarily used to detect trace amounts of o-aminothiophenol and its homologues.

[0008] The determination methods currently retrieved have the following problems: (1) The relative error of determining the content of 2-aminothiophenol by UV-visible spectrophotometry is large, and it is impossible to quantitatively confirm the oxidized impurities; (2) The purity of 2-aminothiophenol is determined by gas chromatography, but the oxidized impurities have a high boiling point and are difficult to vaporize and cannot be detected.

[0009] Therefore, there is an urgent need to establish an accurate and feasible detection method for 2-aminothiophenol-related substances to meet the quality control needs of 2-aminothiophenol and achieve quality control of drugs such as dotinorel and diltiazem. Summary of the Invention

[0010] According to the preparation process of 2-aminothiophenol, the inventors found that 2-aminothiophenol is easily oxidized and deteriorated in the air, and the following impurities are present.

[0011]

[0012] Among them, the impurities 2,2'-dithiodiphenylamine, 1-phenylthiourea, 2-aminobenzothiazole and aniline all contain amino reaction sites, which can be derived and transferred to the raw materials, posing risks to the safety and efficacy of the finished drugs.

[0013] In order to solve the quality control problem of 2-aminothiophenol and thus better control the quality of drugs such as dotinorel and diltiazem, the present invention provides a method for detecting related substances of 2-aminothiophenol, comprising the following steps:

[0014] (1) Preparation of test solution: Take an appropriate amount of 2-aminobenzenethiol, dissolve it in acetonitrile cooled to -5 to 5°C, and dilute it to prepare the test solution;

[0015] (2) Preparation of system suitability solution: Take an appropriate amount of 2-aminothiophenol, add hydrogen peroxide solution, and dilute with acetonitrile to prepare a system suitability solution;

[0016] (3) Chromatographic test: Take the system suitability solution and the test sample solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0017] Furthermore, in step (3), the chromatographic conditions include:

[0018] Chromatographic column: octadecylsilane bonded silica gel or phenyl bonded silica gel as filler;

[0019] Mobile phase: Mobile phase A: phosphate solution-disodium ethylenediaminetetraacetate; Mobile phase B: phosphate solution-disodium ethylenediaminetetraacetate-acetonitrile.

[0020] Furthermore, the phosphate in the mobile phase is selected from one or more of diammonium hydrogen phosphate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate. Preferably, the phosphate is dipotassium hydrogen phosphate.

[0021] Furthermore, the concentration of phosphate in the mobile phase is 5 mmol / L to 30 mmol / L. Preferably, the concentration of phosphate is 5 mmol / L.

[0022] Furthermore, the pH of the phosphate solution in the mobile phase is 5.0 to 7.0. Preferably, the pH of the phosphate solution in the mobile phase is 6.0.

[0023] Furthermore, the concentration of disodium EDTA in the mobile phase is 10 mmol / L to 30 mmol / L. Preferably, the concentration of disodium EDTA is 20 mmol / L.

[0024] Furthermore, in the mobile phase A, the ratio (V / V) of phosphate solution to disodium ethylenediaminetetraacetic acid solution is 85-95:5-15. Preferably, the ratio (V / V) of phosphate solution to disodium ethylenediaminetetraacetic acid solution is 90:10.

[0025] Furthermore, in the mobile phase B, the ratio of phosphate solution to disodium ethylenediaminetetraacetic acid solution to acetonitrile solution (V / V / V) is 15-25:10:65-75. Preferably, the ratio of phosphate solution to disodium ethylenediaminetetraacetic acid solution to acetonitrile solution (V / V / V) is 20:10:70.

[0026] Furthermore, in step (3), the mobile phase gradient elution conditions are as follows:

[0027] .

[0028] Furthermore, in step (3), the mobile phase gradient elution conditions are as follows:

[0029] .

[0030] Furthermore, the chromatographic conditions described in step (3) also include:

[0031] Detector: UV detector;

[0032] Flow rate: 0.5 ml / min to 1.5 ml / min; preferably, the flow rate is 1.0 ml / min;

[0033] Column temperature: 10-40°C; preferably, the column temperature is 20-40°C;

[0034] Detection wavelength: 260nm~290nm; preferably, the detection wavelength is 278nm;

[0035] Injection volume: 10 µl;

[0036] Quantitative method: peak area normalization method.

[0037] The present invention provides a method for detecting 2-aminothiophenol-related substances, which can detect impurities contained in 2-aminothiophenol. The impurities are selected from one or more of 2,2'-dithiodiphenylamine, 1-phenylthiourea, 2-aminobenzothiazole, and aniline.

[0038] The method for detecting 2-aminothiophenol-related substances provided by the present invention has good applicability and is suitable for the quality control of 2-aminothiophenol, and is also suitable for the quality control of dotinorex and diltiazem.

[0039] Beneficial effects

[0040] The inventors discovered that 2-aminothiophenol contains a thiol group and is easily complexed with metal ions in the wall of a chromatographic column, thereby deteriorating the peak shape of the main peak. The present invention uses a chromatographic column filled with octadecylsilane bonded silica gel or phenyl bonded silica gel, and uses dipotassium hydrogen phosphate as mobile phase A and a mixed solution of dipotassium hydrogen phosphate and acetonitrile as mobile phase B. A certain concentration of a metal chelator, disodium ethylenediaminetetraacetate, is added to mobile phases A and B to shield metal ions and optimize the peak shape of 2-aminothiophenol.

[0041] The inventors discovered that using acetonitrile cooled to -5 to 2°C as the solvent for sample preparation improves the step peaks caused by the continuous oxidation of the main component and optimizes the peak shapes of 2-aminothiophenol and the oxidized impurity 2,2'-dithiodiphenylamine. This also addresses the issue of non-reproducible detection of the oxidized impurity 2,2'-dithiodiphenylamine in room temperature solvents, and the method offers excellent precision.

[0042] The present invention does not require a reference substance, and 2,2'-disulfadiphenylamine is degraded through simple material operations to serve as a system applicability solution for 2,2'-disulfadiphenylamine positioning, with strong specificity and low cost.

[0043] The present invention solves the deficiencies in the prior art and provides a reverse-phase HPLC determination method, which can realize the inspection of related substances of 2-aminothiophenol. The method has good specificity, linearity, precision, sensitivity, accuracy and durability. The impurity 2,2'-dithiodiphenylamine is located through an oxidation destruction test, and accurate positioning and quantification of 2,2'-dithiodiphenylamine are achieved. The method can be used for quality control of 2-aminothiophenol and meets the quality control requirements of 2-aminothiophenol. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Chromatograms of the test sample solutions of chromatographic columns 1 and 2 were selected for the method for the determination of related substances of 2-aminothiophenol in Example 2.

[0045] Figure 2 Chromatogram of the test solution of chromatographic column 3 selected for the method of selecting 2-aminothiophenol related substances in Example 2.

[0046] Figure 3 Chromatographic column 4 was selected for the method of selecting 2-aminothiophenol related substances in Example 2 for the chromatogram of the test sample solution.

[0047] Figure 4 Select mobile phase A for Example 3 2-aminothiophenol related substances method and select the chromatogram of the test solution.

[0048] Figure 5 Select mobile phase A for Example 4 2-aminothiophenol related substances method and select the chromatogram of the test solution.

[0049] Figure 6 This is an overlapping diagram of the precision test of the related substances inspection of 2-aminothiophenol in Example 4.

[0050] Figure 7 Chromatogram of the test solution prepared in room temperature solvent for Example 5 2-aminothiophenol related substances inspection.

[0051] Figure 8 This is the chromatogram of the blank solvent interference test in Example 6.

[0052] Figure 9 This is the solution test chromatogram of the separation degree of related substances of 2-aminothiophenol in Example 7.

[0053] Figure 10 This is the chromatogram of the system suitability test for checking related substances of 2-aminothiophenol in Example 8.

[0054] Figure 11 This is the linear diagram of related substances of 2-aminothiophenol in Example 9.

[0055] Figure 12 This is the solution chromatogram of the detection limit of 2-aminothiophenol in Example 10.

[0056] Figure 13 This is the solution chromatogram of the detection limits of 1-phenylthiourea, aniline, 2-aminobenzothiazole, and 2,2'-dithiodiphenylamine in Example 10.

[0057] Figure 14 This is the chromatogram of the quantitative limit solution of 2-aminothiophenol in Example 10.

[0058] Figure 15 This is the chromatogram of the quantitative limit solution of 1-phenylthiourea, aniline, 2-aminobenzothiazole, and 2,2'-dithiodiphenylamine in Example 10.

[0059] Figure 16 This is an overlay diagram of the precision test of related substances of 2-aminothiophenol in Example 11.

[0060] Figure 17 This is an overlay diagram of the test solution in Example 12 at a column temperature of 20-30°C.

[0061] Figure 18 This is the chromatogram of the test solution in Example 12 at a column temperature of 45°C.

[0062] Figure 19 This is an overlapping diagram of the test sample solution test at different solvent temperatures in Example 12.

[0063] Figure 20 The chromatograms of the test sample solutions at different mobile phase pH values ​​in Example 12 (pH 4.6) are shown.

[0064] Figure 21 The chromatograms of the test sample solutions at different mobile phase pH values ​​in Example 12 (pH 7.5) are shown. DETAILED DESCRIPTION

[0065] To facilitate those skilled in the art to understand the contents of the present invention, specific embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0066] Example 1 Preparation of solution

[0067] Preparation of test solution: Take 30 mg of 2-aminobenzenethiol, place it in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0068] Preparation of system suitability solution: Take about 30 mg of 2-aminothiophenol and place it in a 25 ml volumetric flask. Add 0.2 ml of 30% hydrogen peroxide solution, dilute to the mark with acetonitrile, shake well, and let it stand at room temperature for 20 minutes.

[0069] Preparation of impurity stock solutions: Take appropriate amounts of 2,2'-dithiodiphenylamine, 1-phenylthiourea, 2-aminobenzothiazole, and aniline, dissolve them in acetonitrile, and dilute them to make solutions containing approximately 0.6 mg of each impurity per 1 ml, which serve as the impurity stock solutions.

[0070] Preparation of the resolution solution: Take an appropriate amount of 2-aminothiophenol and the stock solutions of each impurity, dissolve them in acetonitrile and dilute them to make a solution containing about 1.2 mg of 2-aminothiophenol and about 1.2 μg of each impurity per 1 ml, which is used as the resolution solution.

[0071] Example 2 Selection of chromatographic conditions

[0072] Chromatographic column 1: Phenyl bonded silica gel as filler

[0073] Column 2: Octadecylsilane bonded silica gel as filler

[0074] Column 3: Octylsilane bonded silica gel as filler

[0075] Column 4: biphenyl bonded silica gel as filler

[0076] Take 10 μl of the test solution prepared according to Example 1, inject it into the liquid chromatograph under different chromatographic column conditions, and record the chromatogram.

[0077] Results: Under chromatographic columns 1 and 2, the peak shapes of the main component and the degradation impurity 2,2'-disulfadiphenylamine were good, and there was no interference peak near the two peaks. The method was specific. See the attached Figure 1 , under chromatographic columns 3 and 4, the peak shape of the degradation impurity 2,2'-disulfadiphenylamine is poor, see attached Figure 2 , Attachment Figure 3 Therefore, the chromatographic column is determined to use phenyl bonded silica gel as the filler or octadecylsilane bonded silica gel as the filler.

[0078] Example 3 Mobile phase selection

[0079] Mobile phase A: 10 mmol / L potassium hydrogen phosphate solution (adjust pH to 6.0 with phosphoric acid)

[0080] Mobile phase B: acetonitrile

[0081] Gradient Table

[0082] .

[0083] Take 10 μl of the test solution prepared according to Example 1, inject it into the liquid chromatograph, and record the chromatogram.

[0084] Results: 2-aminothiophenol peak retention and peak shape were good. When multiple injections were made under the conditions of the column and mobile phase, the peak shape of 2-aminothiophenol deteriorated and a shoulder peak appeared. After flushing with acidic mobile phase, the peak shape improved. When multiple injections were made continuously, the peak shape still deteriorated. See attached. Figure 4 , adding metal chelating agents to the mobile phase can improve the peak shape of 2-aminothiophenol.

[0085] Example 4 Mobile phase selection

[0086] Screening was performed based on the following chromatographic conditions:

[0087] Mobile phase A: 5 mmol / L dipotassium hydrogen phosphate solution (adjust pH to 6.0 with phosphoric acid)-20 mmol / L disodium ethylenediaminetetraacetic acid solution (90:10)

[0088] Mobile phase B: 5 mmol / L potassium hydrogen phosphate solution (adjust pH to 6.0 with phosphoric acid)-20 mmol / L disodium ethylenediaminetetraacetic acid solution-acetonitrile (20:10:70)

[0089] Gradient Table

[0090] .

[0091] Take 10µl of the test solution, inject it into the liquid chromatograph, and record the chromatogram.

[0092] Results: The retention time of the main peak was 12.232 minutes, and the retention time of the impurity 2,2'-dithiodiphenylamine peak was 28.021 minutes. The peak shapes of the two chromatographic peaks were good. Figure 5 Continuous injection of multiple needles, the peak shape is still good, see attached Figure 6 .

[0093] Example 5 Solvent Selection

[0094] 2-Aminothiophenol is sensitive to air and easily oxidized. Under room temperature, the test solution was prepared. The oxidation process in the solution was ongoing, causing a step peak to appear between 2-aminothiophenol and the oxidized impurity 2,2'-dithiodiphenylamine. See attached Figure 7 , and the test solution was freshly prepared before use, the reproducibility of the detected amount of oxidized impurities was still poor. Therefore, a pure organic solvent without water was used to dissolve the sample, and the organic solvent was placed in a low-temperature environment in advance. That is, acetonitrile cooled to -5 to 2°C was used as the solvent for sample preparation. The results showed that the detected amount of oxidized impurities was lower than that detected in the solvent at room temperature, and the reproducibility was good. The results are shown in Table 1.

[0095] Table 1 Test results of 2-aminothiophenol related substances at different solvent temperatures

[0096] .

[0097] Example 6 Solvent Interference Test

[0098] Accurately measure 10 μl of solvent, inject it into the liquid chromatograph, and record the chromatogram. Figure 8 The blank solvent elutes at retention times of 2.585 minutes and 64.323 minutes, respectively, and does not interfere with the determination of related substances of this product.

[0099] Example 7 Specificity Test-Separation Test

[0100] Take 10 μl of the separation solution and the positioning solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The minimum separation between 2-aminothiophenol and the adjacent impurity peaks and between each impurity peak is 5.34, which is good separation. There is no interfering peak near 2,2'-dithiodiphenylamine. The specific results are shown in Table 2 and Appendix. Figure 9 .

[0101] Table 2 Peak positioning and separation test results

[0102] .

[0103] Example 8 System suitability test

[0104] Preparation of system suitability test solution: Weigh an appropriate amount of 2-aminothiophenol, add 0.8% of the final volume of 30% hydrogen peroxide solution, dilute with acetonitrile to make a solution containing approximately 1.2 mg per 1 ml, shake well, and let it stand at room temperature for 20 minutes.

[0105] Take 10μl of the system suitability solution and inject it into the liquid chromatograph, record the chromatogram, and see the attached results. Figure 10 .

[0106] Results: In the system suitability solution chromatogram, the retention time of the main peak was 12.093 minutes, and the retention time of 2,2'-disulfadiphenylamine was 32.577 minutes. The peaks had good shapes and were easy to identify, indicating good method specificity.

[0107] Example 9 Linearity Test

[0108] 2-Aminothiophenol linear stock solution: Accurately weigh approximately 30 mg of 2-aminothiophenol and place it in a 25 ml volumetric flask. Dilute to the mark with acetonitrile (-5 to 2°C) and shake well to prepare the linear stock solution.

[0109] Accurately measure 0.1 ml, 0.2 ml, and 1 ml respectively and place them into three different 10 ml volumetric flasks. Dilute to the scale with acetonitrile (-5 to 2°C), shake well, and use them as linear 1, linear 2, and linear 3 solutions respectively.

[0110] Accurately weigh approximately 15 mg and 36 mg of 2-aminothiophenol respectively, place them into two different 25 ml volumetric flasks, dilute to the scale with acetonitrile (-5 ~ 2 ° C), shake well, and use them as linear 4 and linear 5 solutions respectively.

[0111] Quantitation limit solution: Accurately weigh about 30 mg of 2-aminothiophenol, place it in a 25 ml volumetric flask, dilute it to the scale with acetonitrile (-5 to 2 ° C), accurately measure 0.15 ml, place it in a 100 ml volumetric flask, dilute it to the scale with acetonitrile (-5 to 2 ° C), and shake well.

[0112] Accurately measure 10 μl of each of the above series of solutions and inject them into the liquid chromatograph. Record the chromatogram and plot the peak area versus concentration. Calculate the regression equation and correlation coefficient using the least squares method. The results are shown in Table 3 and Appendix. Figure 11 As shown in the figure, combined with the results of the limit of quantitation test, the linearity of the related substances of 2-aminothiophenol is good.

[0113] Table 3 Linearity test results of 2-aminothiophenol related substances

[0114] .

[0115] Example 10 Limit of Quantitation and Limit of Detection Test

[0116] Take an appropriate amount of each compound and dilute it with acetonitrile cooled to -5-2°C to prepare solutions of different concentrations. Accurately measure 10 μl of each solution and inject it into the liquid chromatograph. Record the chromatogram until the main peak response value is no less than 10 times and 3 times the noise signal. This is the limit of quantification and detection limit of each compound. The results are shown in Tables 4 and 5 and the attached Figures 12-15 .

[0117] Table 4 Detection limit results of 2-aminothiophenol related substances

[0118]

[0119] Table 5 Quantitation limits of related substances of 2-aminothiophenol

[0120] .

[0121] Example 11 Precision Test

[0122] Preparation of test solution: Prepare freshly before use. Weigh an appropriate amount of 2-aminothiophenol and dilute it with acetonitrile cooled to -5-2°C to a solution containing approximately 1.2 mg per 1 ml. This will serve as the test solution. Prepare six parallel aliquots of the test solution.

[0123] Accurately measure 10 μl of each test solution and inject it into the liquid chromatograph to record the chromatogram. The results are shown in Table 6 and Appendix. Figure 16 The results of 6 measurements showed that the detection amount of 2,2'-disulfadiphenylamine was basically consistent (1.28%~1.30%), the detection amount of other largest single impurities was consistent (0.10%~0.11%), and the detection amount of total impurities was consistent (1.46%~1.49%). The results showed that the precision test results were good.

[0124] Table 6 Precision test results

[0125] .

[0126] Example 12 Durability Test

[0127] Accurately measure 10 μl of each test solution and inject them into the liquid chromatograph respectively. Record the chromatogram. Based on the established chromatographic conditions, use different column temperatures, different solvent temperatures, and different pH values ​​of mobile phases according to Table 7 to determine the related substances of this product. The results are as follows: Figures 17-21As shown, when the column temperature was in the range of 20 to 40°C, the solvent temperature was in the range of -5 to 5°C, and the pH value of the mobile phase was in the range of 5.0 to 7.0, the peak shape of 2-2'-disulfadiphenylamine was good, and the detection amounts of 2-2'-disulfadiphenylamine, other largest single impurities, and total impurities were calculated respectively by the peak area normalization method. The impurity spectra were basically the same, the determination results were basically consistent, and the method had good durability.

[0128] At a column temperature of 45°C and mobile phase pH values ​​of 4.6 and 7.5, the peak shape of 2-2'-disulfadiphenylamine was poor, and the method was not applicable.

[0129] Table 7 Durability of the test method for related substances of 2-aminothiophenol

[0130] .

Claims

1. A method for detecting related substances of 2-aminothiophenol, characterized in that: The following steps are involved: (1) Preparation of test solution: Take an appropriate amount of 2-aminothiophenol, dissolve it in acetonitrile cooled to -5 to 2°C, and dilute it to prepare the test solution; (2) Preparation of system suitability solution: Take an appropriate amount of 2-aminothiophenol, add hydrogen peroxide solution, and dilute with acetonitrile to prepare a system suitability solution; (3) Chromatographic test: Take the system suitability solution and the test solution, inject them into the liquid chromatograph respectively, and record the chromatogram; Wherein, the chromatographic conditions in step (3) include: Chromatographic column: octadecylsilane bonded silica gel or phenyl bonded silica gel as filler; Mobile phase: Mobile phase A: phosphate solution-disodium ethylenediaminetetraacetate; Mobile phase B: phosphate solution-disodium ethylenediaminetetraacetate-acetonitrile.

2. The method for detecting related substances of 2-aminothiophenol according to claim 1, wherein The phosphate in the mobile phase is selected from one or more of diammonium hydrogen phosphate, dipotassium hydrogen phosphate and disodium hydrogen phosphate.

3. The method for detecting related substances of 2-aminothiophenol according to claim 2, wherein The phosphate is dipotassium hydrogen phosphate.

4. The method for detecting related substances of 2-aminothiophenol according to claim 1, wherein In the mobile phase A, the volume ratio of phosphate solution to disodium ethylenediaminetetraacetic acid solution is 85-95:5-15.

5. The method for detecting related substances of 2-aminothiophenol according to claim 1, wherein In the mobile phase B, the volume ratio of phosphate solution-disodium ethylenediaminetetraacetic acid solution-acetonitrile solution is 15-25:10:65-75.

6. The method for detecting related substances of 2-aminothiophenol according to claim 1, wherein The mobile phase gradient elution conditions were as follows: 。 7. The method for detecting related substances of 2-aminothiophenol according to claim 6, wherein The mobile phase gradient elution conditions are as follows: 。 8. The method for detecting related substances of 2-aminothiophenol according to claim 1, wherein The chromatographic conditions described in step (3) also include: Detector: UV detector; Flow rate: 0.5ml / min~1.5ml / min; Column temperature: 10-40°C; Detection wavelength: 260nm~290nm; Injection volume: 10 µl; Quantitative method: peak area normalization method.

9. The method for detecting 2-aminothiophenol-related substances according to any one of claims 1 to 8 can detect impurities contained in 2-aminothiophenol, wherein the impurities are selected from one or more of 2,2'-dithiodiphenylamine, 1-phenylthiourea, 2-aminobenzothiazole, and aniline.

Images