High performance liquid chromatography detection method for 4-aminophenol-2, 6-dicarboxylic acid in mesalazine raw material medicine

Abstract

The invention relates to a high performance liquid chromatography detection method for 4-aminophenol-2, 6-dicarboxylic acid in a mesalazine raw material medicine, and belongs to the field of medicine analysis. According to the high performance liquid chromatography detection method for the 4-aminophenol-2, 6-dicarboxylic acid in the mesalazine bulk drug, octadecylsilane chemically bonded silica is adopted as a filling agent, a 0.01-0.06 mol / L phosphate buffer solution is adopted as a mobile phase A, a combination of the mobile phase A and one or two of acetonitrile or methanol is adopted as a mobile phase B, and the content of the 4-aminophenol-2, 6-dicarboxylic acid in the mesalazine bulk drug is detected through the high performance liquid chromatography. Wherein the pH value of the phosphate buffer solution is adjusted to 5.5-7.5 by using a dilute sodium hydroxide solution. The method is accurate and reliable in determination result, the solvent does not interfere with impurity detection, the specificity is good, and the product quality can be well controlled.

Description

Technical Field

[0001] The invention relates to a high performance liquid chromatography detection method for 4-aminophenol-2,6-dicarboxylic acid in a mesalazine raw material drug, and belongs to the field of drug analysis. Background Art

[0002] Mesalazine, also known as 5-aminosalicylic acid, is a nonsteroidal anti-inflammatory drug used to treat acute attacks of ulcerative colitis and Crohn's disease (Crohn's disease). As a synthetic drug, mesalazine may produce process impurities such as 4-aminophenol-2,6-dicarboxylic acid during the synthesis of raw materials. Information on mesalazine and 4-aminophenol-2,6-dicarboxylic acid is shown in Table 1.

[0003] Table 1: Information table of mesalazine and 4-aminophenol-2,6-dicarboxylic acid

[0004]

[0005] There is currently no literature or patent disclosure on the detection of 4-aminophenol-2,6-dicarboxylic acid in mesalazine API by high performance liquid chromatography. In the prior art, the current edition of the United States Pharmacopoeia, the Chinese patent application entitled "A method for detecting specific impurities in mesalazine" and the publication number "CN108169344A", the Chinese patent application entitled "A method for determining mesalazine-related substances by high performance liquid chromatography" and the publication number "CN112630325A", and the Chinese patent application entitled "A method for determining related substances in mesalazine enema" and the publication number "CN109799292A", all of the methods published add ion pair reagents to the mobile phase. The system has poor reproducibility and will lead to chromatographic column modification, which is not conducive to the life cycle management of the method. In addition. The European Pharmacopoeia uses phosphate buffer as the mobile phase. When using this method to detect 4-aminophenol-2,6-dicarboxylic acid, the peak is not completely separated from the solvent peak, and the specificity is poor.

[0006] In order to better and more accurately control the 4-aminophenol-2,6-dicarboxylic acid impurities in the product and ensure the quality of the drug, it is necessary to study an analytical detection method suitable for 4-aminophenol-2,6-dicarboxylic acid in mesalazine raw materials. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a high performance liquid chromatography detection method for 4-aminophenol-2,6-dicarboxylic acid in a mesalazine raw material drug.

[0008] The technical solution of the present invention to solve the technical problem is as follows:

[0009] A high performance liquid chromatography method for detecting 4-aminophenol-2,6-dicarboxylic acid in a mesalazine raw material drug, wherein the detection conditions of the method are as follows: using a reverse phase chromatographic column, the mobile phase comprises a mobile phase A and a mobile phase B, and gradient elution is performed according to the elution procedure in the following table, with the total volume of the mobile phase being 100%;

[0010]

[0011]

[0012] The mobile phase A is a phosphate buffer with a concentration of 0.01 to 0.06 mol / L and a pH value of 5.5 to 7.5; the mobile phase B is a combination of the mobile phase A and acetonitrile or methanol or both; the flow rate of the mobile phase is 0.5 to 2.0 ml / min; the chromatographic column uses octadecylsilane bonded silica gel as filler; the column temperature of the chromatographic column is 10 to 60° C.; the temperature of the injector is 1 to 15° C.;

[0013] The detection process of the method is as follows: accurately measure the system suitability solution, the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram; calculate the content of 4-aminophenol-2,6-dicarboxylic acid by the external standard method.

[0014] Furthermore, the calculation method is to calculate the amount of 4-aminophenol-2,6-dicarboxylic acid by peak area using an external standard method, and the calculation formula is as follows:

[0015]

[0016] Wherein, Mstd is the sample weight of 4-aminophenol-2,6-dicarboxylic acid reference substance, mg; Vstd is the dilution multiple of 4-aminophenol-2,6-dicarboxylic acid reference substance solution; Astd is the chromatographic analysis of the reference substance solution. Figure 4 -aminophenol-2,6-dicarboxylic acid peak area; P is the purity of the reference substance, %; Mt is the sample weight of the test substance, mg; Vt is the dilution factor of the test solution; At is the chromatographic Figure 4 -Aminophenol-2,6-dicarboxylic acid peak area.

[0017] Furthermore, the phosphate buffer is a sodium phosphate buffer, a potassium phosphate buffer or an ammonium phosphate buffer; in some preferred embodiments, the phosphate buffer is a sodium phosphate buffer.

[0018] Furthermore, the concentration of the phosphate buffer is 0.05 mol / L. The pH value of the phosphate buffer is 5.8. The pH value of the phosphate buffer is adjusted with a dilute sodium hydroxide solution, and the concentration of the dilute sodium hydroxide solution is 5.0% to 10.0%; in some preferred embodiments, the concentration of the dilute sodium hydroxide solution is 8.5%.

[0019] Furthermore, the mobile phase B is mobile phase A-acetonitrile in a volume ratio of 70:30.

[0020] Furthermore, the flow rate of the mobile phase is 0.7-1.5 ml / min; in some more preferred embodiments, the flow rate of the mobile phase is 1.0 ml / min.

[0021] Furthermore, the chromatographic column is Waters X-Terra MS C18, with specifications of 4.6 mm×250 mm and 5 μm.

[0022] Further, the analysis method is carried out on a high performance liquid chromatograph, using a differential refractive index detector, an evaporative light scattering detector, an electrospray detector or an ultraviolet detector; in some preferred embodiments, the analysis method is carried out on a high performance liquid chromatograph, using an ultraviolet detector, and the detection wavelength is 240nm. The column temperature of the chromatographic column is 30 to 50°C; in some more preferred embodiments, the column temperature of the chromatographic column is 40°C. The temperature of the injector is 2 to 8°C; in some more preferred embodiments, the temperature of the injector is 5°C.

[0023] In the detection method of the present invention, the preparation method of the detection solution involved is as follows:

[0024] Solvent preparation method: Take 8.5ml of hydrochloric acid, put it in 100ml of water, measure 10ml, and dilute it to 1000ml with water;

[0025] The system suitability solution is prepared as follows: take the reference substance 4-aminophenol-2,6-dicarboxylic acid and dissolve it with a solvent to prepare a solution containing 5 μg of 4-aminophenol-2,6-dicarboxylic acid per 1 ml, and take the reference substance 3-aminosalicylic acid and dissolve it with a solvent to prepare a solution containing 10 μg of 3-aminosalicylic acid per 1 ml, as the reference substance stock solution; take about 10 mg of the mesalazine reference substance, accurately add 1 ml of each of the above two reference substance stock solutions, dissolve with a solvent and dilute to the scale, shake well, and use as the system suitability solution;

[0026] The test solution was prepared as follows: Take an appropriate amount of the mesalazine API to be tested, add a solvent to prepare a solution containing 1.0 mg of mesalazine per 1 ml, as the test solution;

[0027] The preparation method of the reference solution is as follows: take an appropriate amount of 4-aminophenol-2,6-dicarboxylic acid reference substance, dissolve it in a solvent and quantitatively dilute it to make a solution containing 0.5 μg of 4-aminophenol-2,6-dicarboxylic acid per 1 ml, which is used as the reference solution.

[0028] In the most preferred embodiment of the present invention, the HPLC detection method of 4-aminophenol-2,6-dicarboxylic acid in the mesalazine raw material drug, the chromatographic conditions are as follows:

[0029] The analytical method is carried out on a high performance liquid chromatograph; the detector is an ultraviolet detector with a detection wavelength of 240 nm;

[0030] The chromatographic column was an octadecylsilane bonded silica gel column Waters X-TerraMS C18, with specifications of 4.6 mm × 250 mm, 5 μm; the column temperature was 40 °C; the injection volume was 20 μl; and the injector temperature was 5 °C;

[0031] Perform gradient elution according to the following table:

[0032]

[0033] The mobile phase A is sodium phosphate buffer, and the mobile phase B is mobile phase A-acetonitrile: sodium phosphate buffer with a volume ratio of 70:30, a concentration of 0.05 mol / L, and a pH value of 5.8±0.05;

[0034] The flow rate of the mobile phase was 1.0 ml per minute;

[0035] The detection process is as follows:

[0036] Accurately measure the system suitability solution, test solution and reference solution, inject them into the high performance liquid chromatograph respectively, and record the chromatogram; calculate the amount of 4-aminophenol-2,6-dicarboxylic acid by the peak area according to the external standard method:

[0037]

[0038] Wherein, Mstd is the sample weight of 4-aminophenol-2,6-dicarboxylic acid reference substance, mg; Vstd is the dilution multiple of 4-aminophenol-2,6-dicarboxylic acid reference substance solution; Astd is the chromatographic analysis of the reference substance solution. Figure 4 -aminophenol-2,6-dicarboxylic acid peak area; P is the purity of the reference substance, %; Mt is the sample weight of the test substance, mg; Vt is the dilution factor of the test solution; At is the chromatographic Figure 4 -Aminophenol-2,6-dicarboxylic acid peak area.

[0039] If there are impurity peaks in the chromatogram of the test solution, the content of 4-aminophenol-2,6-dicarboxylic acid should not exceed 0.05%.

[0040] Furthermore, the sodium phosphate buffer solution is prepared as follows: sodium dihydrogen phosphate monohydrate is taken and dissolved in water to a concentration of 0.05 mol / L, and the pH value is adjusted to 5.8±0.05 with 8.5% dilute sodium hydroxide solution, and filtered.

[0041] The main key technical means adopted by the present invention to solve the technical problems and the technical effects brought about by them are specifically as follows:

[0042] 1. In the detection method of the present invention, the inventors used a highly selective Waters X-Terra MS C18 (4.6mm×250mm, 5μm) chromatographic column for separation, selected a highly sensitive detection wavelength of 240nm, and used a weakly acidic phosphate-acetonitrile system to keep the chromatographic peak of 4-aminophenol-2,6-dicarboxylic acid in a good peak shape, and used gradient elution to detect 4-aminophenol-2,6-dicarboxylic acid in the product. By using a highly selective Waters X-Terra MS C18 (4.6mm×250mm, 5μm) chromatographic column for separation, 4-aminophenol-2,6-dicarboxylic acid can be well separated from mesalamine and its impurities, avoiding interference. The ultraviolet spectrum of 4-aminophenol-2,6-dicarboxylic acid has a large absorption at a wavelength of 240nm, so using 240nm as the detection wavelength can improve the detection sensitivity of 4-aminophenol-2,6-dicarboxylic acid, and the chromatogram has no obvious gradient peak at this wavelength. By using a weakly acidic phosphate (pH 5.8)-acetonitrile system, 4-aminophenol-2,6-dicarboxylic acid can maintain a good peak shape and good separation from other impurities. By using a specific gradient elution program to detect 4-aminophenol-2,6-dicarboxylic acid of this product, mesalamine and its impurities can be avoided from remaining and interfering with the detection of 4-aminophenol-2,6-dicarboxylic acid. By using a solvent (preparation method: take 8.5 ml of hydrochloric acid, put it in 100 ml of water, measure 10 ml, and dilute it to 1000 ml with water) to dissolve the sample, 4-aminophenol-2,6-dicarboxylic acid can maintain a good peak shape.

[0043] 2. The detection method of the present invention is accurate, simple to operate, has good reproducibility, good sensitivity and specificity, can fully meet the detection of 4-aminophenol-2,6-dicarboxylic acid in this product, ensures product quality, and has strong practicality in work. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 HPLC chromatogram of the system suitability solution.

[0045] Figure 2 HPLC chromatogram of the test solution.

[0046] Figure 3 HPLC chromatogram of the reference solution.

[0047] Figure 4 This is the sensitive solution HPLC chromatogram of 4-aminophenol-2,6-dicarboxylic acid.

[0048] Figure 5This is the linear standard curve of 4-aminophenol-2,6-dicarboxylic acid. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0050] Table 2 Test reference substances

[0051]

[0052] Example 1

[0053] 1. Chromatographic conditions and system suitability experiments:

[0054] 1.1 Chromatographic conditions

[0055] Instrument: High performance liquid chromatography, UV detector.

[0056] For the determination of 4-aminophenol-2,6-dicarboxylic acid in mesalazine, a Waters X-Terra MS C18 (4.6mm×250mm, 5μm) chromatographic column with high chemical stability and good sensitivity was selected. A phosphate buffer-acetonitrile system was used as the mobile phase to improve the buffering effect. Mobile phase A was phosphate buffer [preparation method: take 6.9g of sodium dihydrogen phosphate monohydrate, dissolve it in 1000ml of water, adjust the pH value to 5.8±0.05 with dilute sodium hydroxide solution (8.5g→100mL), and filter], and mobile phase B was mobile phase A-acetonitrile (volume ratio of 70:30). 4-Aminophenol-2,6-dicarboxylic acid has a large absorption at 240nm, and there is no obvious gradient peak in the chromatogram at this wavelength, so this is used as the detection wavelength. The column temperature is 40℃, the flow rate is 1.0ml per minute, the injection volume is 20μl, and the injection temperature is 5℃. After a series of gradient program optimizations, the specific gradient program is as follows:

[0057]

[0058] Under the chromatographic conditions, the peak shape of 4-aminophenol-2,6-dicarboxylic acid was good and it could be well separated from the mesalazine peak and other impurity peaks.

[0059] 1.2 Detection solution

[0060] 1.2.1 Solvent

[0061] Preparation method: Take 8.5 ml of hydrochloric acid, place it in 100 ml of water, measure 10 ml, and dilute it to 1000 ml with water.

[0062] 1.2.2 System suitability solutions

[0063] Preparation method: Take the reference substance 4-aminophenol-2,6-dicarboxylic acid and dissolve it in a solvent to prepare a solution containing about 5 μg of 4-aminophenol-2,6-dicarboxylic acid per 1 ml. Take the reference substance 3-aminosalicylic acid and dissolve it in a solvent to prepare a solution containing about 10 μg of 3-aminosalicylic acid per 1 ml as the reference substance stock solution.

[0064] Take about 10 mg of mesalazine reference substance, place it in a 10 ml volumetric flask, accurately add 1 ml of each of the two reference substance stock solutions mentioned above, dissolve and dilute to the scale with solvent, shake well, and use this as the system suitability solution.

[0065] 1.2.3 API test solution

[0066] Preparation method: Take an appropriate amount of the mesalazine raw material to be tested, add solvent to make a solution containing about 1.0 mg of mesalazine per 1 ml, as the test solution.

[0067] 1.2.4 Reference solution

[0068] Preparation method: Take an appropriate amount of 4-aminophenol-2,6-dicarboxylic acid reference substance, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 0.5 μg of 4-aminophenol-2,6-dicarboxylic acid per 1 ml, which is used as the reference substance solution.

[0069] 1.3 Detection of 4-aminophenol-2,6-dicarboxylic acid in mesalamine

[0070] Accurately measure 20 μl of system suitability solution, test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. The system suitability solution chromatogram is shown in Figure 1 , the chromatogram of the test solution is shown in Figure 2 , the chromatogram of the reference solution is shown in Figure 3 , calculate the amount of 4-aminophenol-2,6-dicarboxylic acid by peak area according to the external standard method:

[0071]

[0072] Wherein, Mstd is the sample weight of 4-aminophenol-2,6-dicarboxylic acid reference substance, mg; Vstd is the dilution factor of 4-aminophenol-2,6-dicarboxylic acid reference substance solution; Astd is the peak area of ​​4-aminophenol-2,6-dicarboxylic acid in the chromatogram of the reference substance solution; P is the purity of the reference substance, %; Mt is the sample weight of the test substance, mg; Vt is the dilution factor of the test substance solution; At is the peak area of ​​4-aminophenol-2,6-dicarboxylic acid in the chromatogram of the test substance solution.

[0073] If there is a 4-aminophenol-2,6-dicarboxylic acid peak in the test solution, it should not exceed 0.05%.

[0074] 2. Exclusivity

[0075] The solvent and system suitability solution were prepared according to the method described in Item 1.2 above. 20 μl of each of the two solutions were accurately measured and injected into the liquid chromatograph respectively. The blank solvent had no interference with the detection of 4-aminophenol-2,6-dicarboxylic acid. The separation of each peak in the system suitability solution was greater than 1.5, which met the requirements.

[0076] 3. Limit of Quantitation and Limit of Detection

[0077] Take an appropriate amount of 4-aminophenol-2,6-dicarboxylic acid reference substance and prepare it with solvent to contain 1×10 -4 mg / ml, 5×10 -5 mg / ml concentration of solution, 20 μl was injected respectively to produce a signal of 4-aminophenol-2,6-dicarboxylic acid at least 10 times the baseline noise. -5 The solution with a concentration of mg / ml is the quantitative limit solution, and the minimum detection amount is 1.0ng (S / N≥10). If the concentration of the sample during the test is about 1mg per 1ml, the quantitative limit is 0.005%. See the chromatogram Figure 4 .

[0078] Take an appropriate amount of 4-aminophenol-2,6-dicarboxylic acid reference substance and prepare it with solvent to contain 3×10 -5 mg / ml, 1.5×10 -5 mg / ml concentration of solution, 20 μl was injected respectively to produce a signal of 4-aminophenol-2,6-dicarboxylic acid at least 3 times the baseline noise. -5 The solution with a concentration of mg / ml is the detection limit solution, and the minimum detection amount is 0.3ng (S / N≥3). If the concentration of the sample during inspection is calculated as approximately 1mg per 1ml, the detection limit is 0.002%.

[0079] The impurity content of 4-aminophenol-2,6-dicarboxylic acid of 0.002% and above can be detected, indicating that the detection sensitivity of this method is high and can fully meet the determination requirements of 4-aminophenol-2,6-dicarboxylic acid inspection.

[0080] 4. Linearity and range

[0081] The HPLC determination method of 4-aminophenol-2,6-dicarboxylic acid in mesalazine of the present invention comprises the following steps: taking about 10 mg of a 4-aminophenol-2,6-dicarboxylic acid reference substance, accurately weighing it, placing it in a 50 ml volumetric bottle, dissolving it with a solvent and diluting it to the mark, shaking it well, accurately measuring 5 ml and placing it in a 100 ml volumetric bottle, diluting it to the mark with a solvent, shaking it well, and using it as a reference substance reserve solution. Accurately measuring 2.5 ml, 5 ml, 7.5 ml, 5 ml, and 7.5 ml of the reserve solution respectively and placing them in 200 ml, 200 ml, 200 ml, 100 ml, and 100 ml volumetric bottles, diluting it to the mark with a solvent, shaking it well, and using it as a linear solution. When the concentration of 4-aminophenol-2,6-dicarboxylic acid is in the range of 0.0644 to 0.7733 μg / ml, the peak area and concentration show a good linear relationship, and the linear equation is: y = 31602.2766x + 126.0609, and the correlation coefficient is 1.0000. The linear standard curve is shown in Figure 5 .

[0082] 5. Accuracy

[0083] Take 4-aminophenol-2,6-dicarboxylic acid reference substance and prepare a solution with a concentration of 0.01mg / ml with solvent as the recovery rate stock solution. Take 100mg of the test sample, accurately weigh it, place it in a 100ml volumetric flask, dissolve it with solvent and dilute it to the scale, shake it well, and use it as the background solution. Take 100mg of the test sample, accurately weigh it, place it in a 100ml volumetric flask, and prepare 9 portions, of which 3 portions accurately add 0.5ml of the recovery rate stock solution as the quantitative limit level accuracy solution, 3 portions accurately add 5ml of the recovery rate stock solution as the 100% accuracy solution, and 3 portions accurately add 7.5ml of the recovery rate stock solution as the 150% accuracy solution, respectively dissolve it with solvent and dilute it to the scale, shake it well. Accurately measure 20μl of each solution and inject it into the liquid chromatograph. Calculate the recovery rate according to the background amount, addition amount and detection amount of 4-aminophenol-2,6-dicarboxylic acid in each accuracy solution. The results are shown in Table 3.

[0084] Table 3 Recovery results

[0085]

[0086]

[0087] The recovery rates of 4-aminophenol-2,6-dicarboxylic acid impurity at high, medium and low concentrations were between 95.67% and 103.88%, and the RSD value of the recovery rate was 2.3%. The results showed that the method had good accuracy.

[0088] 6. Repeatability

[0089] According to the method described in Item 1.2 above, prepare system suitability solution and reference solution, prepare 6 portions of test solution in parallel, prepare 6 portions of 100% accuracy solution in parallel as the method under the accuracy item, use them as spiked test solution, and perform sampling and detection according to the chromatographic conditions described in Item 1.1 above. Accurately measure 20 μl of each of system suitability solution, reference solution, test solution and spiked test solution, and inject them into the liquid chromatograph respectively. According to Item 1.3 above, calculate the amount of 4-aminophenol-2,6-dicarboxylic acid by peak area according to the external standard method. No 4-aminophenol-2,6-dicarboxylic acid was detected in the test solution. The average value of 4-aminophenol-2,6-dicarboxylic acid measured in the spiked test solution was 0.05%, and the RSD was 0.3%, indicating that the method has good repeatability.

[0090] 7. Solution stability

[0091] Take one portion each of the reference solution, test solution and 100% accuracy solution, place them in a sample tray at 4°C, and inject and measure them at 0, 6, 12, 18 and 24 hours respectively to investigate the change in the peak area of ​​4-aminophenol-2,6-dicarboxylic acid. The results show that when the above three solutions were placed at 4°C for 24 hours, the RSD of the peak area of ​​the reference solution was 0.9%, 4-aminophenol-2,6-dicarboxylic acid was not detected in the test solutions, and the RSD of the peak area of ​​the 100% accuracy solution was 1.1%, indicating that the method has good solution stability within 24 hours.

[0092] The above experimental results show that the method is simple and sensitive to operate, has good specificity and repeatability, and can better control the quality of mesalazine.

[0093] 8. Sample testing

[0094] Take 3 batches of test samples, prepare system suitability solution, reference solution and test sample solution according to the method described in Item 1.2 above, perform sample injection and detection according to the chromatographic conditions described in Item 1.1 above, accurately measure 20 μl of each of system suitability solution, reference solution and test sample solution, and inject them into the liquid chromatograph respectively, calculate the amount of 4-aminophenol-2,6-dicarboxylic acid by peak area according to the external standard method as described in Item 1.3 above, and the results are shown in Table 4.

[0095] Table 4 Sample test results

[0096]

[0097] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A high performance liquid chromatography detection method for 4-aminophenol-2,6-dicarboxylic acid in a mesalazine bulk drug, characterized in that: The detection conditions of the method are as follows: using a reverse phase chromatographic column, the mobile phase includes mobile phase A and mobile phase B, and gradient elution is performed according to the elution program in the following table, with the total volume of the mobile phase being 100%; The mobile phase A is a phosphate buffer with a concentration of 0.01 to 0.06 mol / L and a pH value of 5.5 to 7.5; the mobile phase B is a combination of the mobile phase A and acetonitrile or methanol or both; the flow rate of the mobile phase is 0.5 to 2.0 ml / min; the chromatographic column uses octadecylsilane bonded silica gel as filler; the column temperature of the chromatographic column is 10 to 60° C.; the temperature of the injector is 1 to 15° C.; The detection process of the method is as follows: accurately measure the system suitability solution, the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram; calculate the content of 4-aminophenol-2,6-dicarboxylic acid by the external standard method.

2. The high performance liquid chromatography detection method according to claim 1, characterized in that: The calculation method is to calculate the amount of 4-aminophenol-2,6-dicarboxylic acid by peak area using an external standard method, and the calculation formula is as follows: Wherein, Mstd is the sample weight of 4-aminophenol-2,6-dicarboxylic acid reference substance, mg; Vstd is the dilution factor of 4-aminophenol-2,6-dicarboxylic acid reference substance solution; Astd is the peak area of ​​4-aminophenol-2,6-dicarboxylic acid in the chromatogram of the reference substance solution; P is the purity of the reference substance, %; Mt is the sample weight of the test substance, mg; Vt is the dilution factor of the test substance solution; At is the peak area of ​​4-aminophenol-2,6-dicarboxylic acid in the chromatogram of the test substance solution.

3. The high performance liquid chromatography detection method according to claim 1, characterized in that: The phosphate buffer is sodium phosphate buffer, potassium phosphate buffer or ammonium phosphate buffer.

4. The high performance liquid chromatography detection method according to claim 3, characterized in that: The concentration of the phosphate buffer is 0.05 mol / L, and the pH value of the phosphate buffer is 5.8; the pH value of the phosphate buffer is adjusted by a dilute sodium hydroxide solution, and the concentration of the dilute sodium hydroxide solution is 5.0% to 10.0%.

5. The high performance liquid chromatography detection method according to claim 1, characterized in that: The mobile phase B is mobile phase A-acetonitrile in a volume ratio of 70:

30.

6. The high performance liquid chromatography detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.7-1.5 ml / min.

7. The high performance liquid chromatography detection method according to claim 1, characterized in that: The chromatographic column is Waters X-Terra MS C18, with specifications of 4.6 mm×250 mm and 5 μm.

8. The high performance liquid chromatography detection method according to claim 1, characterized in that: The analysis method is carried out on a high performance liquid chromatograph, using a differential refractive index detector, an evaporative light scattering detector, an electrospray detector or an ultraviolet detector; the detection wavelength is 240nm; the column temperature of the chromatographic column is 30-50°C; and the temperature of the injector is 2-8°C.

9. The high performance liquid chromatography detection method according to any one of claims 1 to 8, characterized in that: The detection method and chromatographic conditions are as follows: The analytical method is carried out on a high performance liquid chromatograph; the detector is an ultraviolet detector with a detection wavelength of 240 nm; The chromatographic column was an octadecylsilane bonded silica gel column Waters X-TerraMS C18, with specifications of 4.6 mm × 250 mm, 5 μm; the column temperature was 40 °C; the injection volume was 20 μl; and the injector temperature was 5 °C; Perform gradient elution according to the following table: The mobile phase A is sodium phosphate buffer, and the mobile phase B is mobile phase A-acetonitrile: sodium phosphate buffer with a volume ratio of 70:30, a concentration of 0.05 mol / L, and a pH value of 5.8±0.05; The flow rate of the mobile phase was 1.0 ml per minute; The detection process is as follows: Accurately measure the system suitability solution, test solution and reference solution, inject them into the high performance liquid chromatograph respectively, and record the chromatogram; calculate the amount of 4-aminophenol-2,6-dicarboxylic acid by the peak area according to the external standard method: Wherein, Mstd is the sample weight of 4-aminophenol-2,6-dicarboxylic acid reference substance, mg; Vstd is the dilution factor of 4-aminophenol-2,6-dicarboxylic acid reference substance solution; Astd is the peak area of ​​4-aminophenol-2,6-dicarboxylic acid in the chromatogram of the reference substance solution; P is the purity of the reference substance, %; Mt is the sample weight of the test substance, mg; Vt is the dilution factor of the test substance solution; At is the peak area of ​​4-aminophenol-2,6-dicarboxylic acid in the chromatogram of the test substance solution.

10. The high performance liquid chromatography detection method according to claim 9, characterized in that: The sodium phosphate buffer solution is prepared as follows: sodium dihydrogen phosphate monohydrate is dissolved in water to a concentration of 0.05 mol / L, the pH value is adjusted to 5.8±0.05 with 8.5% dilute sodium hydroxide solution, and filtered.

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