A method for detecting the content of genotoxic impurities in indobufen raw materials

Abstract

The present invention discloses a method for detecting the content of genotoxic impurities in indobufen raw materials, which is used to detect that the genotoxic impurity is 2-(4-hydroxyaminophenyl)butyric acid, and relates to the field of pharmaceutical analytical chemistry. The analytical method adopts high performance liquid chromatography, and the chromatographic conditions are: chromatographic column: pentafluorophenyl bonded silica gel column or chromatographic column with equivalent performance to the chromatographic column, mobile phase A: phosphate buffer, pH value adjusted to 2.0-3.0 with phosphoric acid, mobile phase B: mobile phase A-methanol, mobile phases A and B are carried out according to a gradient program; column temperature: 35°C-45°C; flow rate: 0.4-0.6 ml / min; detection wavelength: 240 nm; injection volume: 20 μl. The method realizes the detection of genotoxic impurities in indobufen raw materials. The method has good specificity, high detection sensitivity and accuracy, and low detection cost, which is of great significance for achieving quality control and safety assurance of indobufen raw materials.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical analytical chemistry, and specifically relates to a detection method for determining genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials, which is used for the analysis and limit determination of genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials. Background Art

[0002] Indobufen is an inhibitor of platelet aggregation. Its active ingredient is an isoindolinylbutyric acid derivative, chemically named (±) 2-[4-(1-oxo-2-isoindolinyl)phenyl]butyric acid, with a molecular formula of C 18 H 17 NO3, molecular weight is 295.34, structural formula is:

[0003]

[0004] As an anticoagulant and antithrombotic agent, indobufen can inhibit the release of platelet factors such as ADP, 5-hydroxytryptamine, platelet factor 4, and β-thromboglobulin, thereby playing an antiplatelet aggregation role. This product does not affect the plasma parameters of blood coagulation, but only prolongs the bleeding time. Therefore, after achieving the therapeutic purpose, the drug can be quickly discontinued and the abnormal platelet function can be restored to normal. This product is non-mutagenic, non-teratogenic, and non-toxic to the fetus. It is used for arteriosclerotic ischemic vascular lesions, ischemic cerebrovascular lesions and peripheral arterial lesions, dyslipidemia, venous thrombosis and diabetes. It can also be used to prevent thrombosis during extracorporeal circulation surgery.

[0005] Most of the reported synthetic processes of indobufen involve 2-(4-hydroxyaminophenyl)butyric acid. Patent CN106631974B discloses the following synthetic process:

[0006] (1) Hydrogenation reaction

[0007]

[0008] (2) Cyclization reaction

[0009]

[0010] (3) Zinc powder reduction reaction

[0011]

[0012] This process route uses 2-(4-nitrophenyl)butyric acid as the starting material to obtain the key intermediate 2-(4-aminophenyl)butyric acid. During the reaction, 2-(4-hydroxyaminophenyl)butyric acid is generated, which contains a genotoxic warning structure and is a genotoxic impurity. There is a risk of being transferred to the indobufen API. The structural formula of the genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid is:

[0013]

[0014] According to the relevant provisions of the ICH M7 guidelines and combined with the maximum daily dose of indobufen tablets (indobufen 400 mg), the maximum daily intake of 2-(4-hydroxyaminophenyl)butyric acid according to the TTC method shall not exceed 1.5 μg. Therefore, the control limit of 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials shall not exceed 3.75 ppm. This limit is relatively low and requires the detection method to have high sensitivity and specificity. Currently, there is no literature reporting the detection method of this impurity.

[0015] The quality standards for indobufen are not included in domestic and foreign pharmacopoeias. In the relevant substance inspection of the national drug standard [WS1-(X-069)-2006Z-2021], only the method for controlling unknown single impurities and total impurities is announced. However, this method is not suitable for the detection of the known impurity 2-(4-hydroxyaminophenyl)butyric acid. The sensitivity of the method does not meet the requirements and cannot effectively detect the content of 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials.

[0016] Patent document CN 202011171014.1 discloses a method for detecting genotoxic impurities in indobufen API, which uses high performance liquid chromatography-mass spectrometry to separate and determine the genotoxic impurities 2-(4-amino)phenylbutyric acid and / or 2-(4-nitro)phenylbutyric acid, using octadecylsilane bonded silica gel or octylsilane bonded silica gel as the stationary phase, and a mixture of solvent A and solvent B as the mobile phase, wherein solvent A is selected from one or a mixed aqueous solution of ammonium formate or ammonium acetate, and solvent B is selected from any one containing methanol, acetonitrile or a mixture of the two. This method requires the use of a mass spectrometer detector with an electrospray ion source, and the detection cost is high, and this detection method is not suitable for the detection of the genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid in indobufen API.

[0017] Patent document CN 202011399490.9 discloses a method for detecting indobufen impurities, which uses an ultraviolet detector for analysis and detection, uses octadecylsilane bonded silica gel or octyl bonded silica gel as the stationary phase for chromatographic separation, and the mobile phase is a mixture of solvent A and solvent B, wherein solvent A is an aqueous solution of potassium dihydrogen phosphate, and solvent B is selected from methanol, acetonitrile, or a mixture of the two. However, when this method is used for the detection of the genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid of the present invention, the detection of 2-(4-hydroxyaminophenyl)butyric acid and the detection of 2-(4-aminophenyl)butyric acid cannot be completely separated, and the degree of separation is less than 1.5 required by the methodology, and the impurity 2-(4-hydroxyaminophenyl)butyric acid cannot be accurately quantitatively analyzed. Based on this, the present invention hopes to provide a detection method that can accurately detect the content of the genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid in the indobufen raw material.

[0018] At present, there is no public literature or patent reporting on the detection method of 2-(4-hydroxyaminophenyl)butyric acid, a genotoxic impurity in indobufen. Therefore, finding a highly sensitive and specific detection method to control the genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid in indobufen API is of great significance to improving product quality and drug safety. Summary of the invention

[0019] In order to solve the above problems, the purpose of the present invention is to provide a method for detecting the content of 2-(4-hydroxymethylaminophenyl)butyric acid, a genotoxic impurity, in indobufen raw materials, which can effectively achieve the determination and limit control of 2-(4-hydroxymethylaminophenyl)butyric acid, a genotoxic impurity in indobufen raw materials. The detection method has high sensitivity, strong specificity, good accuracy, and is simple and fast to operate.

[0020] To achieve the above object, the technical solution of the present invention is as follows:

[0021] A method for detecting the content of genotoxic impurities in indobufen raw materials, the method adopts high performance liquid chromatography detection, and the chromatographic conditions are:

[0022] Chromatographic column: pentafluorophenyl bonded silica gel chromatographic column;

[0023] Mobile phase A: 0.05 mol / L phosphate buffer, pH adjusted to 2.5 with phosphoric acid;

[0024] Mobile phase B: mobile phase A-methanol;

[0025] Column temperature: 35℃-45℃;

[0026] Flow rate: 0.4 ml / min~0.6 ml / min;

[0027] Detection wavelength: 240 nm;

[0028] Injection volume: 20 μl;

[0029] Mobile phases A and B were run according to a gradient program.

[0030] Among them, the preferred chromatographic column is Welch Ultimate PFP, 250 mm×4.6 mm, 5µm or a chromatographic column with equivalent performance to the chromatographic column.

[0031] Preferably, mobile phase A is 0.05 mol / L phosphate buffer, the pH value of mobile phase A is adjusted to 2.0-3.0 with phosphoric acid, the phosphate is any one or both of potassium dihydrogen phosphate and sodium dihydrogen phosphate, and in mobile phase B, the volume ratio of mobile phase A to methanol is 15-25:85-75.

[0032] The preferred flow rate is 0.5 ml / min;

[0033] The preferred column temperature is 40°C;

[0034] The preferred detection wavelength is 240 nm;

[0035] The injection gradient elution program was:

[0036] Time / minute Mobile phase A / % Mobile phase B / % 0 85 15 25 35 65 35 10 90 55 10 90 56 85 15 80 85 15

[0037] The percentages of mobile phase A and B are volume percentages.

[0038] This method can be used to detect the content of genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials, thereby performing quality control of the 2-(4-hydroxyaminophenyl)butyric acid content in indobufen raw materials.

[0039] The beneficial effects of the present invention are:

[0040] 1) The present invention is suitable for detecting the content of 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials, filling the gap in the method for detecting the genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials, and is helpful for quantitative analysis and control of impurities in the preparation process of indobufen raw materials.

[0041] 2) The present invention provides a method for detecting the content of 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials by using high performance liquid chromatography technology, which has low detection limit, strong method specificity, good repeatability and high sensitivity.

[0042] 3) A known amount of 2-(4-hydroxyaminophenyl)butyric acid reference substance was added to the indobufen API sample, and the recovery rate was measured to be 99.50%, indicating good accuracy of the method.

[0043] In addition, the method of the present invention can realize detection by using ordinary high performance liquid chromatography, without the need to purchase and connect other instruments, and is simple and easy to operate, and has strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the chromatogram of the blank solvent of 2-(4-hydroxyaminophenyl)butyric acid in Example 1.

[0045] Figure 2 This is the chromatogram of the quantification limit solution of 2-(4-hydroxyaminophenyl)butyric acid in Example 1.

[0046] Figure 3 This is the peak result of the quantification limit solution of 2-(4-hydroxyaminophenyl)butyric acid in Example 1.

[0047] Figure 4 This is the chromatogram of the detection limit solution of 2-(4-hydroxyaminophenyl)butyric acid in Example 1.

[0048] Figure 5 This is the peak result of the 2-(4-hydroxyaminophenyl)butyric acid detection limit solution in Example 1.

[0049] Figure 6 This is the chromatogram of the 2-(4-hydroxyaminophenyl)butyric acid reference solution in Example 1.

[0050] Figure 7 This is the chromatogram of the 2-(4-hydroxyaminophenyl)butyric acid test solution in Example 1.

[0051] Figure 8 This is the chromatogram of 2-(4-hydroxyaminophenyl)butyric acid detection at a column temperature of 30° C. in Example 3.

[0052] Fig. 9 This is the chromatogram of 2-(4-hydroxyaminophenyl)butyric acid detection at a column temperature of 45° C. in Example 3.

[0053] Fig.10 This is the detection chromatogram of 2-(4-hydroxyaminophenyl)butyric acid at a flow rate of 0.4 ml / min in Example 4.

[0054] Fig.11 This is the detection chromatogram of 2-(4-hydroxyaminophenyl)butyric acid at a flow rate of 0.6 ml / min in Example 4.

[0055] Fig.12 This is the chromatogram of 2-(4-hydroxyaminophenyl)butyric acid detection when the pH of the buffer salt in the mobile phase is 2.0 in Example 5.

[0056] Fig.13This is the chromatogram of 2-(4-hydroxyaminophenyl)butyric acid detection when the pH of the buffer salt in the mobile phase is 3.0 in Example 5.

[0057] Fig.14 This is the chromatogram when the concentration of the 2-(4-hydroxyaminophenyl)butyric acid reference substance in Comparative Example 1 is 0.075 μg / ml.

[0058] Fig.15 This is the chromatogram when the concentration of the 2-(4-hydroxyaminophenyl)butyric acid reference substance in Comparative Example 2 is 1.5 μg / ml. DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to the accompanying drawings and in combination with specific embodiments. The embodiments are provided to better illustrate the content of the present invention, but the content of the present invention is not limited to the embodiments. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above content, which still fall within the scope of protection of the present invention.

[0060] Sources of reference substances involved in the examples:

[0061] Genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid, Jinan Kanghe Pharmaceutical Technology Co., Ltd., purity 97.84%, batch number IPA210511-01.

[0062] The indobufen API was selected from the following three batches:

[0063] Batches 20220501, 20220502 and 20220601, the manufacturer is Shandong Lusheng Pharmaceutical Co., Ltd.

[0064] Example 1 Methodological Investigation of the Detection Method of 2-(4-Hydroxyaminophenyl)butyric Acid in Indobufen

[0065] With reference to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 9101, Guidelines for Validation of Analytical Methods - Impurity Limits, the specificity, limit of quantification, limit of detection and accuracy of this method were investigated;

[0066] (1) Chromatographic conditions

[0067] Instrument: Agilent 1260 high performance liquid chromatograph;

[0068] Column: Welch Ultimate PFP, 250 mm × 4.6 mm, 5 μm;

[0069] Mobile phase A: 0.05 mol / L phosphate buffer, pH adjusted to 2.5 with phosphoric acid;

[0070] The volume ratio of mobile phase B: mobile phase A-methanol was 20:80;

[0071] Elution was performed according to the following gradient:

[0072] Time / minute Mobile phase A / % Mobile phase B / % 0 85 15 25 35 65 35 10 90 55 10 90 56 85 15 80 85 15

[0073] Among them, the percentages of mobile phase A and B are volume percentages;

[0074] Flow rate: 0.5 ml / min;

[0075] Column temperature: 40°C;

[0076] Detection wavelength: 240 nm;

[0077] Injection volume: 20 μl;

[0078] (2) Solution preparation

[0079] Proprietary solution preparation

[0080] Step (1) Take an appropriate amount of 2-(4-hydroxyaminophenyl)butyric acid impurity reference substance, accurately weigh it, add a solvent (acetonitrile-water (75:25)) to dissolve it and dilute it to make a solution containing 0.75 μg per 1 ml; use it as the impurity reference substance stock solution;

[0081] Step (2) Accurately measure 1 ml of the solution in step (1), place it in a 25 ml volumetric flask, and dilute it to the mark with solvent; this serves as the reference solution;

[0082] Step (3) Take an appropriate amount of indobufen raw material, accurately weigh it, dissolve it in a solvent and dilute it to prepare a solution containing 8 mg of indobufen per 1 ml, as the test solution;

[0083] Step (4) Take an appropriate amount of indobufen API, accurately weigh it, add an appropriate amount of the solution from step (1), dissolve it with a solvent and dilute it to prepare a mixed solution containing 8 mg of indobufen and 0.03 μg of 2-(4-hydroxyaminophenyl)butyric acid per 1 ml, as the spiked test solution;

[0084] Preparation of Quantitation Limit and Detection Limit Solutions

[0085] Step (1) Take an appropriate amount of 2-(4-hydroxyaminophenyl)butyric acid impurity reference substance, weigh it accurately, add solvent (acetonitrile-water (75:25)) to dissolve and dilute to make a solution containing about 0.75 μg per 1 ml; use it as the impurity reference substance stock solution;

[0086] Step (2) taking an appropriate amount of the solution in step (1) and diluting it step by step with a solvent to a concentration where the signal-to-noise ratio S / N≈10 and S / N≈3;

[0087] Accuracy solution preparation

[0088] Step (1) Take an appropriate amount of 2-(4-hydroxyaminophenyl)butyric acid impurity reference substance, weigh it accurately, add solvent (acetonitrile-water (75:25)) to dissolve and dilute to make a solution containing 0.75 μg per 1 ml; use it as the impurity reference substance stock solution;

[0089] Step (2) Take 1 ml of the solution in step (1), place it in a 25 ml volumetric flask, dilute it to the mark with solvent, and shake well; this is used as the impurity reference solution;

[0090] Step (3) Take 200 mg of indobufen API, place in a 25 ml volumetric flask, dissolve and dilute to scale with solvent, shake well, and use as the test solution;

[0091] Step (4) Take 200 mg of indobufen API, place it in a 25 ml volumetric flask, add 1 ml of the solution from step (1), dissolve it with a solvent and dilute it to the mark, shake well, and use this as the recovery solution (prepare 6 portions in the same way);

[0092] Sample test solution preparation

[0093] Step (1) Take an appropriate amount of 2-(4-hydroxyaminophenyl)butyric acid impurity reference substance, weigh it accurately, add solvent (acetonitrile-water (75:25)) to dissolve and dilute to make a solution containing 0.75 μg per 1 ml; use it as the impurity reference substance stock solution;

[0094] Step (2) Accurately measure 1 ml of the solution in step (1), place it in a 25 ml volumetric flask, and dilute it to the mark with solvent; this serves as the reference solution;

[0095] Step (3) Take an appropriate amount of indobufen raw material, accurately weigh it, dissolve it in a solvent and dilute it to make a solution containing 8 mg per 1 ml, which is used as the test solution;

[0096] (3) Determination method

[0097] Specificity determination method: accurately measure 20 μl of blank solvent, impurity reference solution, test solution and mixed solution respectively, inject them into liquid chromatograph respectively, and record the chromatogram. Blank solvent, test solution and other impurities should not interfere with the detection of 2-(4-hydroxyaminophenyl)butyric acid;

[0098] Determination method of quantification limit and detection limit: measure the quantification limit and detection limit solutions respectively. When the signal-to-noise ratio is about 3, the corresponding concentration is the detection limit. When the signal-to-noise ratio is about 10, the corresponding concentration is the quantification limit.

[0099] Recovery rate determination method: accurately measure 20 μl of blank solvent, impurity reference solution, test solution, and recovery rate solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. The individual recovery rate and average value of 2-(4-hydroxyaminophenyl)butyric acid should be between 80% and 115%.

[0100] Determination of 2-(4-hydroxyaminophenyl)butyric acid in samples: Precisely measure blank solvent, 6 portions of impurity reference solution and test solution, inject them into liquid chromatograph, and record the chromatogram. Calculate the residual amount of 2-(4-hydroxyaminophenyl)butyric acid in the sample by peak area according to the external standard method.

[0101] (4) Measurement results

[0102] Specificity: Under this condition, the blank solvent and the test solution had no interference with the detection of 2-(4-hydroxyaminophenyl)butyric acid, and the method had good specificity.

[0103] The quantitative limit and detection limit test data are shown in Table 1 below:

[0104] Table 1 Quantitation limit and detection limit test data of 2-(4-hydroxyaminophenyl)butyric acid

[0105] project Concentration / μg / ml Average S / N Equivalent to main component / ppm Equivalent to limit / % Limit of Quantitation 0.003 10.5 0.375 10 Detection limit 0.0009 3.3 0.1125 3

[0106] The recovery rate test data is shown in Table 2 below:

[0107] Table 2 2-(4-hydroxyaminophenyl)butyric acid recovery test data

[0108] concentration Original amount / μg Addition amount / μg Measured amount / μg Recovery rate / % 100% 0.000 0.736 0.728 98.91 100% 0.000 0.736 0.726 98.64 100% 0.000 0.736 0.735 99.86 100% 0.000 0.736 0.742 100.8 100% 0.000 0.736 0.731 99.32 100% 0.000 0.736 0.732 99.46

[0109] The test data of 3 batches of samples are shown in Table 3 below:

[0110] Table 3 Test data of 2-(4-hydroxyaminophenyl)butyric acid in 3 batches of indobufen raw material samples

[0111] Genotoxic impurities Limit requirements 20220501 20220502 20220601 2-(4-Hydroxyaminophenyl)butyric acid ≤3.75 ppm Not detected Not detected Not detected

[0112] The results of methodological investigation and sample determination show that this method has good specificity, high sensitivity and good accuracy, and can be used to detect the genotoxic impurity 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials. The quantitative limit and detection limit test spectra are shown in the attached Figures 1 to 5 , Typical reference substance solution spectrum is attached Figure 6 , See the attached sample solution spectrum Figure 7 .

[0113] Example 2 Robustness of the method

[0114] (1) Chromatographic conditions: Except for the chromatographic column, other chromatographic conditions were the same as those in Example 1. The chromatographic column was GL Sciences InertSustain PFP (250 mm × 4.6 mm, 5 μm).

[0115] (2) Solution preparation and determination methods are the same as in Example 1

[0116] (3) Sampling and result analysis

[0117] 20 μl of blank solvent, reference solution, test solution and spiked test solution were accurately measured, injected into the liquid chromatograph, and the chromatogram was recorded. Results The blank and test solutions had no interference with the detection of 2-(4-hydroxyaminophenyl)butyric acid. The content of 2-(4-hydroxyaminophenyl)butyric acid in the test solution (see Table 4 below) and the content of 2-(4-hydroxyaminophenyl)butyric acid in the spiked test solution (see Table 5 below) were both the same as the results of Example 1, indicating that the chromatographic column and method are feasible.

[0118] Table 4 Test data of 2-(4-hydroxyaminophenyl)butyric acid in 3 batches of indobufen raw material samples.

[0119] Genotoxic impurities Limit requirements 20220501 20220502 20220601 2-(4-Hydroxyaminophenyl)butyric acid ≤3.75 ppm Not detected Not detected Not detected

[0120] Table 5 Comparison of test data of different chromatographic columns.

[0121] Genotoxic impurities Column 1Welch Ultimate PFP, 250 mm × 4.6 mm, 5 µm Column 2GL SciencesInertSustain PFP, 250 mm × 4.6 mm, 5 μm 2-(4-Hydroxyaminophenyl)butyric acid content 3.74 ppm 3.72 ppm

[0122] Example 3 A method for detecting genotoxic impurities in indobufen - determination of column temperature

[0123] (1) Chromatographic conditions: Except for the column temperature, other chromatographic conditions were the same as those in Example 1. The column temperatures were set to 30°C, 40°C, and 45°C, respectively.

[0124] (2) The preparation and determination methods of the spiked test solution are the same as those in Example 1;

[0125] (3) Sampling and result analysis

[0126] Accurately measure 20 μl of each spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. The results show that when the column temperature is set to 30°C, 40°C, and 45°C, the separation between 2-(4-hydroxyaminophenyl)butyric acid and adjacent peaks at different temperatures meets the requirements, and the determination results of the spiked test solution are consistent (see Table 6). Considering the durability of the method, the chromatographic column temperature of the method is selected to be 40°C.

[0127] Table 6 Comparison of detection data at different column temperatures

[0128] Column temperature 30℃ 40℃ 45℃ 2-(4-Hydroxyaminophenyl)butyric acid content 3.70 ppm 3.74 ppm 3.73 ppm

[0129] Example 4 A method for detecting genotoxic impurities in indobufen - determination of flow rate

[0130] (1) Chromatographic conditions: Except for the flow rate, other chromatographic conditions were the same as those in Example 1, and the flow rates were set to 0.4 ml / min, 0.5 ml / min, and 0.6 ml / min, respectively;

[0131] (2) The preparation and determination methods of the spiked test solution are the same as those in Example 1;

[0132] (3) Sampling and result analysis

[0133] Accurately measure 20 μl of the spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. The test results are shown in Table 6. The results show that when the flow rate is set to 0.4 ml / min, 0.5 ml / min, and 0.6 ml / min, the separation between 2-(4-hydroxyaminophenyl)butyric acid and adjacent peaks meets the requirements, and the determination results are consistent (see Table 7). Considering the durability of the method, the flow rate of the method is selected as 0.5 ml / min.

[0134] Table 7 Comparison of test data at different flow rates

[0135] Flow rate 0.4 ml / min 0.5 ml / min 0.6 ml / min 2-(4-Hydroxyaminophenyl)butyric acid content 3.72 ppm 3.74 ppm 3.75 ppm

[0136] Example 5 A method for detecting genotoxic impurities in indobufen - determination of pH value of buffer salt in mobile phase

[0137] (1) Chromatographic conditions: Except for the pH value of the phosphate buffer in mobile phase A, the other chromatographic conditions were the same as those in Example 1. Phosphoric acid was used to adjust the pH values ​​of the buffer salt in mobile phase A to 2.0, 2.5, and 3.0, respectively.

[0138] (2) The preparation and determination methods of the spiked test solution are the same as those in Example 1;

[0139] (3) Sampling and result analysis

[0140] Accurately measure 20 μl of the spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. The results show that when the pH value of the buffer salt in the mobile phase is 2.0, 2.5, and 3.0, the separation degree of 2-(4-hydroxyaminophenyl)butyric acid and the adjacent peaks meets the requirements, and the determination results are consistent (see Table 8). Considering the durability of the method, the pH of the mobile phase is selected as 2.5.

[0141] Table 8 Comparison of detection data of mobile phase A at different pH values.

[0142] pH value of phosphate buffer in mobile phase A 2.0 2.5 3.0 2-(4-Hydroxyaminophenyl)butyric acid content 3.69 ppm 3.74 ppm 3.72 ppm

[0143] Example 6 A method for detecting genotoxic impurities in indobufen - different volume ratios of mobile phase A and methanol in mobile phase B.

[0144] (1) Chromatographic conditions: The chromatographic conditions were the same as those in Example 1 except that the volume ratio of mobile phase A to methanol in mobile phase B was different. The volume ratios of mobile phase A to methanol in mobile phase B were 15:85 and 25:75, respectively.

[0145] (2) Solution preparation and determination methods are the same as in Example 1

[0146] (3) Sampling and result analysis

[0147] 20 μl of blank solvent, reference solution, test solution and spiked test solution were accurately measured and injected into the liquid chromatograph to record the chromatogram. Results The blank and test solutions had no interference with the detection of 2-(4-hydroxyaminophenyl)butyric acid. The content of 2-(4-hydroxyaminophenyl)butyric acid in the spiked test solution (see Table 9 below) was the same as that in Example 1, indicating that the chromatographic column and method were feasible.

[0148] Table 9 Comparison of detection data of different volume ratios of mobile phase A-methanol in mobile phase B

[0149] Different volume ratios of mobile phase A and methanol in mobile phase B 15:85 20:80 25:75 2-(4-Hydroxyaminophenyl)butyric acid content 3.71 ppm 3.74 ppm 3.72 ppm

[0150] Comparative test of different chromatographic columns

[0151] Comparative Example 1

[0152] (1) Chromatographic conditions: Except for the chromatographic column, other chromatographic conditions were the same as those in Example 1. The chromatographic column was GL Inertsil ODS-3 (150 mm × 4.6 mm, 3 μm);

[0153] (2) Preparation of reference solution

[0154] Step (1) Take an appropriate amount of 2-(4-hydroxyaminophenyl)butyric acid impurity reference substance, accurately weigh it, add a solvent (acetonitrile-water (75:25)) to dissolve it and dilute it to make a solution containing 0.75 μg per 1 ml; use it as the impurity reference substance stock solution;

[0155] Step (2) Accurately measure 1 ml of the solution in step (1), place it in a 10 ml volumetric flask, and dilute it to the mark with solvent to make a solution containing 0.075 μg per 1 ml; this is used as the reference solution;

[0156] (3) Sampling and result analysis

[0157] Accurately measure 20 μl of the reference solution, inject it into the liquid chromatograph, and record the chromatogram. The results show that when the reference concentration is 0.075 μg / ml, there is no peak of 2-(4-hydroxyaminophenyl)butyric acid in the chromatogram. See the attached detection spectrum Fig.14 .

[0158] The results showed that the detection ability of GL Inertsil ODS-3 (150 mm×4.6 mm, 3μm) column was much lower than that of Welch Ultimate PFP (250 mm×4.6 mm, 5μm) column when other chromatographic conditions were the same, and it was not suitable for the detection of 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials.

[0159] Comparative Example 2

[0160] (1) Chromatographic conditions: Except for the chromatographic column, other chromatographic conditions were the same as those in Example 1. The chromatographic column was GL Inertsil ODS-3 (150 mm × 4.6 mm, 3 μm);

[0161] (2) Preparation of reference solution

[0162] Take an appropriate amount of 2-(4-hydroxyaminophenyl)butyric acid impurity reference substance, weigh accurately, dissolve in solvent (acetonitrile-water (75:25)) and dilute to make a solution containing 1.5 μg per 1 ml; use it as the reference substance solution;

[0163] (3) Sampling and result analysis

[0164] Accurately measure 20 μl of the above reference solution, inject it into the liquid chromatograph, and record the chromatogram. The results show that when the reference concentration is 1.5 μg / ml, the peak shape of 2-(4-hydroxyaminophenyl)butyric acid in the chromatogram is poor and the response is low. The detection spectrum is attached. Fig.15 .

[0165] The results showed that GL Inertsil ODS-3 (150 mm×4.6 mm, 3μm) column was not suitable for the detection of 2-(4-hydroxyaminophenyl)butyric acid in indobufen raw materials.

[0166] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention in detail rather than to limit it. Professional and technical personnel in the field can modify or replace the technical solution of the present invention with equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for detecting the content of genotoxic impurities in indobufen bulk drug, characterized in that: The genotoxic impurity is 2-(4-hydroxyaminophenyl)butyric acid; The method is high performance liquid chromatography, and the chromatographic conditions are: Chromatographic column: pentafluorophenyl bonded silica gel column, 250mm×4.6mm, 5μm; Column temperature: 40°C; Flow rate: 0.5ml / min; Detection wavelength: 240nm; Injection volume: 20 μl; Mobile phase A: phosphate buffer, pH adjusted to 2.5 with phosphoric acid; Mobile phase B: mobile phase A-methanol; Mobile phases A and B were carried out according to the gradient program, and the elution program was: The percentages of mobile phase A and B are volume percentages.

2. The method according to claim 1, characterized in that The chromatographic column is any one of Welch Ultimate PFP, 250 mm×4.6 mm, 5 µm and GL Sciences InertSustain PFP, 250 mm×4.6 mm, 5 μm.

3. The method according to claim 1, characterized in that The mobile phase A: the concentration of the phosphate buffer is 0.05 mol / L, and the pH value is adjusted to 2.5 with phosphoric acid; the phosphate is either potassium dihydrogen phosphate or sodium dihydrogen phosphate or both.

4. The method according to claim 1, characterized in that The mobile phase B is a mixed solution of mobile phase A and methanol, and the volume ratio of mobile phase A to methanol is 15-25:85-75.

5. The method according to claim 1, characterized in that: The mobile phase B is a mixed solution of mobile phase A and methanol, and the volume ratio of mobile phase A to methanol is 20:80.

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