Method for determining content of bis (2-aminophenyl) disulfide in 2-aminothiophenol

Abstract

The invention discloses a method for determining the content of bis (2-aminophenyl) disulfide in 2-aminothiophenol. The method comprises the following steps: preparing an acetonitrile solution of citric acid as a diluent; preparing a system applicability solution, a reference solution and a test solution by using a diluent; a diluent, a system applicability solution, a reference substance solution and a test solution are sequentially injected by adopting a liquid chromatography, and the content of bis (2-aminophenyl) disulfide in a 2-aminothiophenol sample is determined by adopting an external standard method. The antioxidant citric acid is added into the diluent, so that the generation of the disulfide compound is inhibited, the existing disulfide compound is not damaged, the solution stability is high, the operation is simple, the system applicability, specificity, accuracy, precision and durability completely accord with the guidance principle verified by the Chinese pharmacopoeia method, and the method is suitable for industrial production. The method can be used for quality control of 2-aminothiophenol.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis methods, and particularly to a method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol. Background Art

[0002] 2-aminothiophenol, also known as o-aminothiophenol, 2-aminothiophenol, and 2-aminobenzenethiol, is a pale yellow liquid or solid with a foul odor and is insoluble in water. It is an important raw material for medicine and organic synthesis, is used in the dye field, and as an antibiotic, can be used to treat infectious diseases caused by certain bacteria.

[0003] 2-aminothiophenol is the main starting material for the synthesis of dostinex. However, 2-aminothiophenol is extremely prone to conversion into bis(2-aminophenyl) disulfide during storage and use. The presence of this impurity seriously affects the quality of subsequent intermediates and finished products. Therefore, it is necessary to detect and control the above-mentioned impurity. In the prior art, liquid chromatography or gas chromatography is generally used to detect the impurity. However, the detection results of the impurity bis(2-aminophenyl) disulfide are poorly reproducible during the detection by conventional liquid or gas chromatography, and the accuracy of the detection results cannot be guaranteed.

[0004] Patent CN114397393A discloses a method for determining diphenyl disulfide in thiophenol by a liquid phase method. The sample solution diluent is acetonitrile, freshly prepared before use, and the sample tray temperature is 5°C. However, 2-aminothiophenol has an amino group in its structure. Compared with thiophenol, 2-aminothiophenol is more likely to polymerize to form bis(2-aminophenyl) disulfide impurities, and the problem cannot be solved by freshly preparing before use and reducing the sample tray temperature.

[0005] Patent CN113552257A discloses a method for determining isomeric impurities in p-fluorothiophenol by a liquid phase method. This method prevents the formation of disulfide compounds by adding a TCEP solution (tris(2-carboxyethyl)phosphine), so that p-fluorothiophenol and its impurities can exist in the form of single molecules, ensuring the stability of the solution. However, it will also reduce the disulfide compounds generated during the storage and use of the sample into monomers, resulting in distorted detection results of the disulfide compounds. Summary of the Invention

[0006] The present invention aims to overcome the above problems existing in the method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol in the prior art, and provides a method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol. By adding the antioxidant citric acid to the diluent, the generation of disulfide compounds is inhibited, and the original disulfide compounds are not destroyed. The solution has high stability, and the operation is simple. It fully complies with the guiding principles of the method validation of the Chinese Pharmacopoeia in terms of system suitability, specificity, accuracy, precision and durability, and can be used for the quality control of 2-aminothiophenol.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol, comprising the following steps: (1) Prepare an acetonitrile solution of citric acid as a diluent; (2) Dissolve the 2-aminothiophenol reference substance and the bis(2-aminophenyl) disulfide reference substance in the diluent to obtain a system suitability solution; (3) Dissolve the bis(2-aminophenyl) disulfide reference substance in the diluent to obtain a reference substance solution; (4) Dissolve the 2-aminothiophenol sample to be tested in the diluent to obtain a test solution; (5) Inject the diluent, the system suitability solution, the reference substance solution and the test solution into the liquid chromatograph in sequence, and use the external standard method to determine the content of bis(2-aminophenyl) disulfide in the 2-aminothiophenol sample.

[0008] The present invention inhibits the generation of disulfide compounds by adding the antioxidant citric acid to the diluent, and does not destroy the original disulfide compounds, so the solution has high stability. Using the method of the present invention to detect bis(2-aminophenyl) disulfide in 2-aminothiophenol has the advantages of high system suitability, strong specificity, meeting the standards in terms of detection limit and quantitation limit, good linear relationship, good precision, good accuracy and good durability. It is suitable for the detection of related substances of 2-aminothiophenol to effectively control the quality of 2-aminothiophenol. At the same time, the determination method of the present invention is simple in operation and accurate in detection results; the detection method of the present invention complies with the guiding principles of the method validation of the Chinese Pharmacopoeia in terms of system suitability, specificity, accuracy, precision and durability.

[0009] Preferably, in the diluent obtained in step (1), the mass ratio of citric acid to acetonitrile is 9-11:91-89.

[0010] Preferably, in the system suitability solution obtained in step (2), the content of 2-aminothiophenol is 0.9-1.1 mg / mL, and the content of bis(2-aminophenyl) disulfide is 25-35 μg / mL.

[0011] Preferably, in the reference solution obtained in step (3), the content of bis(2-aminophenyl) disulfide is 25 to 35 μg / mL.

[0012] Preferably, in the test solution obtained in step (4), the content of 2-aminothiophenol sample is 0.8-1.2 mg / mL.

[0013] Preferably, in step (5), the mobile phase is used for gradient elution during liquid chromatography detection, and the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of phosphoric acid, and mobile phase B is a mixed solution of methanol and acetonitrile, and the elution gradient is, by volume fraction: 0-5min: mobile phase A 60%, mobile phase B 40%; 5-20min: Mobile phase A from 60% to 10%, mobile phase B from 40% to 90%; 20-35min: mobile phase A 10%, mobile phase B 90%; 35-35.1min: mobile phase A from 10% to 60%, mobile phase B from 90% to 40%; 35.1~40min: mobile phase A 60%, mobile phase B 40%.

[0014] Preferably, in mobile phase A, the mass concentration of phosphoric acid is 0.08-0.12%; in mobile phase B, the volume ratio of methanol to acetonitrile is 48-52:52-48.

[0015] Preferably, the chromatographic conditions for liquid chromatography detection in step (5) are as follows: the chromatographic column is a phenyl column; the flow rate is 1.0±0.1 mL / min; the column temperature is 30°C±5°C; the injection volume is 5 μL; and the detection wavelength is 210-254 nm.

[0016] Preferably, the chromatographic column is Agilent Eclipse plus phenyl-Hexyl, with phenyl bonded silica gel as the filler stationary phase.

[0017] The present invention also provides an application of the above method in the production quality control of 2-aminothiophenol, a starting material of polytinorel.

[0018] Therefore, the present invention has the following beneficial effects: The method of the present invention has high system applicability, strong specificity, meets the standards in terms of detection limit and quantitation limit, has a good linear relationship, good precision, good accuracy, and good durability. It is applicable to the detection of related substances of 2-aminothiophenol to effectively control the quality of 2-aminothiophenol. At the same time, the determination method of the present invention is simple to operate and the detection results are accurate; the detection method of the present invention meets the guiding principles of method verification in the Chinese Pharmacopoeia in terms of system applicability, specificity, accuracy, precision, and durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the liquid chromatogram of the blank solution in Example 1 of the present invention.

[0020] Figure 2 It is the liquid chromatogram of the system suitability solution in Example 1 of the present invention.

[0021] Figure 3 It is the liquid chromatogram of the test solution in Example 1 of the present invention.

[0022] Figure 4 It is the linear relationship diagram of bis(2-aminophenyl) disulfide in Example 1 of the present invention.

[0023] Figure 5 It is the liquid chromatogram of the blank solution in Comparative Example 1 of the present invention.

[0024] Figure 6 It is the liquid chromatogram of the system suitability solution in Comparative Example 1 of the present invention.

[0025] Figure 7 It is the liquid chromatogram of the test solution in Comparative Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0027] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

[0028] In order to further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with specific embodiments. However, it should be understood that these descriptions are only for further explaining the features and advantages of the present invention rather than limiting the claims of the present invention.

[0029] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, the present invention may be practiced in other ways than those specifically described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0030] Secondly, as used herein, "one embodiment" or "an embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that exclude each other.

[0031] Example 1: A method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol, comprising the following steps: (1) Solution preparation: Diluent: Prepare an acetonitrile solution of citric acid as the diluent, and the mass concentration of citric acid in the diluent is 10%.

[0032] Blank solution: Diluent.

[0033] System suitability solution: Take the reference substance of 2-aminothiophenol and the reference substance of bis(2-aminophenyl) disulfide, place them in a volumetric flask, dissolve with the diluent and make up to the mark, shake well to obtain a system suitability solution containing 1.0 mg of 2-aminothiophenol and 30 μg of bis(2-aminophenyl) disulfide per 1 mL.

[0034] Reference solution: Take the reference substance of bis(2-aminophenyl) disulfide, place it in a volumetric flask, dissolve with the diluent and make up to the mark, shake well to obtain a reference solution containing 30 μg of bis(2-aminophenyl) disulfide per 1 mL.

[0035] Test solution: Take the sample of 2-aminothiophenol to be tested, place it in a volumetric flask, dissolve with the diluent and make up to the mark, shake well to obtain a test solution containing 1.0 mg of 2-aminothiophenol per 1 mL.

[0036] Spiked test solution: Take the test sample of 2-aminothiophenol and the reference substance of bis(2-aminophenyl) disulfide, place them in a volumetric flask, dissolve with the diluent and make up to the mark, shake well to obtain a spiked test solution containing 1.0 mg of 2-aminothiophenol and 30 μg of bis(2-aminophenyl) disulfide per 1 mL.

[0037] Linear stock solution: Take 25 mg of the reference substance of bis(2-aminophenyl) disulfide, accurately weigh it, place it in a 25 mL volumetric flask, dissolve with the diluent and dilute to the mark, shake well to obtain a linear stock solution.

[0038] LOD solution: Accurately measure 1.0 mL of the linear stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well; then accurately measure 1.0 mL of the resulting solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well to obtain the LOD solution (0.01%).

[0039] Linear solution 1 (LOQ solution): Accurately measure 1.0 mL of the linear stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well; then accurately measure 3.0 mL of the resulting solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well. Prepare 6 parallel portions (0.03%).

[0040] Linear solution 2 (limit 50%): Accurately measure 1.5 mL of the linear stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well (1.5%).

[0041] Linear solution 3 (limit 80%): Accurately measure 2.4 mL of the linear stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well (2.4%).

[0042] Linear solution 4 (limit 100%): Accurately measure 3.0 mL of the linear stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well (3.0%).

[0043] Linear solution 5 (limit 120%): Accurately measure 3.6 mL of the linear stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well (3.6%).

[0044] Linear solution 6 (limit 150%): Accurately measure 4.5 mL of the linear stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well (4.5%).

[0045] Accuracy solution 1: Accurately weigh 25 mg of the 2-aminothiophenol test substance, place it in a 25 mL volumetric flask, dissolve and dilute it to the mark with the LOQ solution, and shake well. Prepare 3 parallel portions (LOQ).

[0046] Accuracy solution 2: Accurately weigh 25 mg of the 2-aminothiophenol test substance, place it in a 25 mL volumetric flask, dissolve and dilute it to the mark with the linear solution 4, and shake well. Prepare 3 parallel portions (limit 100%).

[0047] Accuracy solution 3: Accurately weigh 25 mg of the 2-aminothiophenol test substance, place it in a 25 mL volumetric flask, dissolve and dilute it to the mark with the linear solution 6, and shake well. Prepare 3 parallel portions (limit 150%).

[0048] (2) Determine the content of bis(2-aminophenyl) disulfide in the 2-aminothiophenol test substance by liquid chromatography: 1) Experimental materials and instrument conditions Instrument: Agilent 1260, chromatographic column: Agilent Eclipse plus phenyl-Hexyl 4.6 mm × 250 mm, 5 μm; flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 5 μL, detection wavelength: 215 nm; mobile phase A: 0.1 wt% phosphoric acid aqueous solution (phosphoric acid is HPLC grade, water is high-purity water), mobile phase B: methanol-acetonitrile (1:1 v / v) (both acetonitrile and methanol are HPLC grade), and the gradient elution program is shown in Table 1.

[0049] Table 1: Gradient elution program. Time (min) 0 5 20 35 35.1 40 Mobile phase A (%) 60 60 10 10 60 60 Mobile phase B (%) 40 40 90 90 40 40

[0050] 2) Injection procedure: After the system is stable, inject 2 needles of blank solution, 1 needle of system suitability solution, 6 needles of reference solution, 1 needle of test solution, 1 needle of spiked test solution, and 1 needle of reference solution at the end of the sequence, record the chromatogram, and the chromatograms of the blank solution, system suitability solution, and test solution are as Figures 1 to 3 shown below.

[0051] 3) Requirements: In the system suitability solution, the number of theoretical plates is calculated based on the peak of bis(2-aminophenyl) disulfide and is not less than 5000, and the resolution between bis(2-aminophenyl) disulfide and the adjacent chromatographic peak is not less than 1.5; for the reference solution, inject 6 needles repeatedly, the RSD of the peak area of the main peak chromatographic peak is not more than 5.0%, and the RSD of the retention time is not more than 1.0%; for 7 needles of reference solution, the RSD of the peak area of the main peak chromatographic peak is not more than 5.0%, and the RSD of the retention time is not more than 1.0%.

[0052] 4) Calculation method: Calculate the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to the following formula:

[0053] In the formula: A 样品 : Peak area of bis(2-aminophenyl) disulfide in the test solution; A 对照 : Average value of the peak area of bis(2-aminophenyl) disulfide in 6 needles of reference solution; C 对照 : Concentration of bis(2-aminophenyl) disulfide in the reference solution, mg / mL; C 样品 : Concentration of the test solution, mg / mL.

[0054] System suitability test: System suitability is achieved by using the system suitability solution and the reference solution. It is required that the number of theoretical plates calculated based on the bis(2-aminophenyl) disulfide peak in the system suitability solution is not less than 5000, and the resolution between bis(2-aminophenyl) disulfide and the adjacent chromatographic peak is not less than 1.5. The reference solution is injected 6 times repeatedly, and the RSD of the peak area of the main peak chromatographic peak is not more than 5.0%, and the RSD of the retention time is not more than 1.0%. For the 7-reference solution, the RSD of the peak area of the main peak chromatographic peak is not more than 5.0%, and the RSD of the retention time is not more than 1.0%. The test results are shown in Table 1.

[0055] The specificity of the detection method described in the present invention: Specificity is achieved by detecting the blank solution, the bis(2-aminophenyl) disulfide positioning solution, the test solution, and the spiked test solution. It is required that the blank solution does not interfere with the detection of bis(2-aminophenyl) disulfide; for the bis(2-aminophenyl) disulfide positioning solution, the peak purity of its chromatographic peak is not less than 990; the test solution does not interfere with the detection of bis(2-aminophenyl) disulfide, the resolution between bis(2-aminophenyl) disulfide and the adjacent chromatographic peak is not less than 1.5, and the peak purity of the bis(2-aminophenyl) disulfide chromatographic peak is not less than 990; in the spiked test solution, the detection of bis(2-aminophenyl) disulfide is not interfered, the resolution between bis(2-aminophenyl) disulfide and the adjacent chromatographic peak is not less than 1.5, and the peak purity of the bis(2-aminophenyl) disulfide chromatographic peak is not less than 990. The test results are shown in Table 1.

[0056] The precision of the detection method described in the present invention: Precision is achieved by calculating the RSD of the detection results of the related substance (bis(2-aminophenyl) disulfide) in 2-aminothiophenol in 6 spiked test solutions. It is required that the RSD of the amount of bis(2-aminophenyl) disulfide in the 6 spiked test solutions meets the acceptable standard. After the system is stable, inject 2 needles of the blank solution, 1 needle of the system suitability solution, 6 needles of the reference solution, and 1 needle of each of the 6 spiked test solutions, record the chromatogram, and then inject 1 needle of the reference solution at the end of the sequence. In the 6 spiked test solutions, the RSD of the detection results of bis(2-aminophenyl) disulfide should not be more than 10.0%. The test results are shown in Table 1.

[0057] The detection limit and quantification limit of the detection method described in the present invention: The detection limit was obtained by detecting that the ratio of its response signal to the noise was approximately 3:1, and the quantitation limit was obtained by a signal-to-noise ratio of approximately 10:1. At this concentration level, six replicates of the quantitation limit solution were examined. It was required that the RSD of the peak areas in the six obtained chromatograms should meet the requirements to confirm that the quantitation limit test results had a certain precision. After the system was stable, two injections of the blank solution, one injection of the LOD solution, and one injection of each of the six LOQ solutions were made, and the chromatograms were recorded; the test results are shown in Table 1.

[0058] Linearity and range of the detection method described in the present invention: As Figure 4 shown, within the range of the LOQ concentration to 150% of the limit concentration, six points were taken. With the concentration as the abscissa and the peak area as the ordinate, a linear regression was performed. It was required that the peak area was linear within the range of LOQ to 150% of the limit, and the linear correlation coefficient R 2 met the acceptable standard. After the system was stable, two injections of the linear solution at each concentration were made, and the chromatograms were recorded. Bis(2-aminophenyl) disulfide was linear within the range of LOQ to 150% of the limit concentration, and the linear correlation coefficient R 2 should be not less than 0.995; the test results are shown in Table 1.

[0059] Accuracy of the detection method described in the present invention: Bis(2-aminophenyl) disulfide at three concentrations of LOQ, 100% of the limit, and 150% of the limit was added to the test sample, and its recovery rate was detected. It was required that the recovery rate was within the range of 80.0% - 120.0%, and the RSD of the recovery rate was not more than 10.0%; the test results are shown in Table 1.

[0060] Robustness of the detection method described in the present invention: The law of the change of the detection results with time was investigated after the system suitability solution, reference solution, test sample solution, and spiked test sample solution were placed at room temperature for a period of time, to provide a reference for the placement time of the system suitability solution, test sample solution, and reference solution during detection. After the system was stable, two injections of the blank solution, and one injection of the system suitability solution, reference solution, test sample solution, and spiked test sample solution at different times were made, and the chromatograms were recorded. It was required that in the system suitability solution, the number of theoretical plates calculated based on the peak of bis(2-aminophenyl) disulfide was not less than 5000, and the resolution between bis(2-aminophenyl) disulfide and the adjacent chromatographic peak was not less than 1.5; in the reference solution, the RSD of the peak area of the main peak chromatographic peak was not more than 5.0%, and the RSD of the retention time was not more than 1.0%; in the test sample solution, the RSD of the content of bis(2-aminophenyl) disulfide did not exceed 10.0%; in the spiked test sample solution, the RSD of the content of bis(2-aminophenyl) disulfide was not more than 10.0%; the test results are shown in Table 1.

[0061] Comparative Example 1 (without adding citric acid): A method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol, comprising the following steps: (1) Solution preparation: Diluent: Acetonitrile.

[0062] Blank solution: Diluent.

[0063] System suitability solution: Take the reference substance of 2-aminothiophenol and the reference substance of bis(2-aminophenyl) disulfide, place them in a volumetric flask, dissolve with the diluent and make up to the mark, shake well to obtain a system suitability solution containing 1.0 mg of 2-aminothiophenol and 30 μg of bis(2-aminophenyl) disulfide per 1 mL.

[0064] Reference substance solution: Take the reference substance of bis(2-aminophenyl) disulfide, place it in a volumetric flask, dissolve with the diluent and make up to the mark, shake well to obtain a reference substance solution containing 30 μg of bis(2-aminophenyl) disulfide per 1 mL.

[0065] Test sample solution: Take the 2-aminothiophenol sample to be tested, place it in a volumetric flask, dissolve with the diluent and make up to the mark, shake well to obtain a test sample solution containing 1.0 mg of 2-aminothiophenol per 1 mL.

[0066] (2) Determine the content of bis(2-aminophenyl) disulfide in the 2-aminothiophenol test sample by liquid chromatography: 1) The experimental materials and instrument conditions are the same as those in Example 1.

[0067] 2) Injection procedure: After the system is stable, inject 2 needles of the blank solution, 1 needle of the system suitability solution, 6 needles of the reference substance solution, 1 needle of the test sample solution, and inject 1 needle of the reference substance solution at the end of the sequence, record the chromatogram, and the chromatograms of the blank solution, the system suitability solution, and the test sample solution are as Figures 5 to 7 shown.

[0068] Test the system suitability, specificity, and durability of the method in Comparative Example 1 in the same way as in Example 1, and the results are shown in Table 1.

[0069] Table 1: Determination results.

[0070] As can be seen from the measurement results in Table 1, in Example 1, the method of the present invention is adopted. By adding the antioxidant citric acid to the diluent, the generation of disulfide compounds is inhibited without destroying the original disulfide compounds, and the solution has high stability. Using the method of the present invention to detect bis(2-aminophenyl) disulfide in 2-aminothiophenol has the advantages of high system suitability, strong specificity, meeting the standards in terms of detection limit and quantification limit, good linear relationship, good precision, good accuracy, and good durability. It is applicable to the detection of related substances of 2-aminothiophenol to effectively control the quality of 2-aminothiophenol. At the same time, the measurement method of the present invention is simple to operate and the detection results are accurate. In Comparative Example 1, the antioxidant citric acid is not added to the diluent. Due to the oxidation degradation of 2-aminothiophenol into bis(2-aminophenyl) disulfide, the detection result of bis(2-aminophenyl) disulfide is inaccurate and the system adaptability fails to meet the standard.

[0071] The above are only the preferred embodiments of the present invention and do not limit the present invention in any formal or substantial way. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any equivalent changes such as slight modifications, decorations, and evolutions made by those skilled in the art who are familiar with this specialty without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol, characterized in that: The following steps are involved: (1) Prepare citric acid in acetonitrile solution as diluent; (2) Dissolving 2-aminothiophenol reference substance and bis(2-aminophenyl) disulfide reference substance in a diluent to obtain a system suitability solution; (3) dissolving the bis(2-aminophenyl) disulfide reference substance in a diluent to obtain a reference substance solution; (4) dissolving the 2-aminothiophenol sample to be tested in a diluent to obtain a test solution; (5) The diluent, system suitability solution, reference solution and test solution were injected in sequence by liquid chromatography, and the content of bis(2-aminophenyl) disulfide in the 2-aminothiophenol sample was determined by the external standard method.

2. The method for measuring the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to claim 1, characterized in that: In the diluent obtained in step (1), the mass ratio of citric acid to acetonitrile is 9-11:91-89.

3. The method for measuring the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to claim 1, characterized in that: In the system suitability solution obtained in step (2), the content of 2-aminothiophenol is 0.9-1.1 mg / mL, and the content of bis(2-aminophenyl) disulfide is 25-35 μg / mL.

4. The method for measuring the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to claim 1, characterized in that: In the reference solution obtained in step (3), the content of bis(2-aminophenyl) disulfide is 25-35 μg / mL.

5. The method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to claim 1, characterized in that: In the test solution obtained in step (4), the content of 2-aminothiophenol sample is 0.8~1.2 mg / mL.

6. The method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to claim 1, characterized in that: In step (5), the mobile phase is used for gradient elution during liquid chromatography detection. The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of phosphoric acid, and mobile phase B is a mixed solution of methanol and acetonitrile. The elution gradient is calculated by volume fraction: 0~5min: mobile phase A 60%, mobile phase B 40%; 5-20min: Mobile phase A from 60% to 10%, mobile phase B from 40% to 90%; 20-35min: mobile phase A 10%, mobile phase B 90%; 35-35.1min: mobile phase A from 10% to 60%, mobile phase B from 90% to 40%; 35.1~40min: mobile phase A 60%, mobile phase B 40%.

7. The method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to claim 6, characterized in that: In mobile phase A, the mass concentration of phosphoric acid is 0.08~0.12%; in mobile phase B, the volume ratio of methanol to acetonitrile is 48~52:52~48.

8. The method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to claim 1 or 6, characterized in that: The chromatographic conditions for liquid chromatography detection in step (5) are as follows: chromatographic column is a phenyl column; flow rate: 1.0±0.1mL / min; column temperature is 30℃±5℃; injection volume: 5µL; detection wavelength is 210~254nm.

9. The method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol according to claim 8, characterized in that: The chromatographic column was Agilent Eclipse plus phenyl-Hexyl, with phenyl bonded silica gel as the filler and stationary phase.

10. An application of the method for determining the content of bis(2-aminophenyl) disulfide in 2-aminothiophenol as claimed in any one of claims 1 to 9, characterized in that: Applied to the production quality control of 2-aminothiophenol, the starting material of dotinorel.

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