Detection method for separating and determining related substances in 2, 5-dichlorobenzoic acid

Abstract

The invention provides a detection method for separating and determining various related substances in an initial raw material 2, 5-dichlorobenzoic acid of ixazomib citrate, which adopts a reversed-phase high-performance liquid chromatography with a C18 liquid chromatographic column, adopts a mixed solution of a phosphoric acid solution and acetonitrile as a mobile phase, and adopts a gradient elution method for separating and determining various related substances in the 2, 5-dichlorobenzoic acid as the initial raw material of ixazomib citrate. Gradient elution is carried out by changing the proportion of two solvents in a mobile phase, nine impurities such as various isomers in 2, 5-dichlorobenzoic acid can be effectively separated and measured, the separation degree is good, the peak shape is good, and the quality of an ixazomib citrate raw material medicine can be effectively controlled.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and relates to a method for detecting related substances of a starting material for synthesizing an ixazomib citrate raw material, and specifically relates to a method for separating and determining related substances such as 2,5-dichlorobenzoic acid isomers of the ixazomib citrate starting material. Background Art

[0002] Multiple myeloma is a malignant tumor characterized by abnormal proliferation of plasma cells. Ixazomib citrate is the first new type of targeted drug for the oral treatment of multiple myeloma. It was successfully developed by Japan's Takeda Pharmaceutical Company and approved for marketing by the U.S. FDA in November 2015.

[0003] The chemical name of ixazomib citrate is [2-[(1R)-1-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]-5-oxo-1,3,2-dioxaborolane-4,4-diacetic acid, and the structural formula is as follows:

[0004]

[0005] 2,5-Dichlorobenzoic acid is a key starting material for the synthesis of ixazomib citrate, and its structural formula is:

[0006]

[0007] Chinese patent CN201510680783 "Synthesis of proteasome inhibitor MLN9708" describes a method for synthesizing ixazomib citrate using 2,5-dichlorobenzoic acid as a starting material. The reaction route is as follows:

[0008]

[0009] 2,5-Dichlorobenzoic acid may produce a variety of positional isomers during the synthesis process. Their existence greatly affects the purity of the ixazomib citrate raw material prepared subsequently, affecting the safety of medication.

[0010] According to the synthetic route of 2,5-dichlorobenzoic acid, at least the following nine impurities may be produced during the synthesis of this compound, which may affect the quality of the final product:

[0011]

[0012] Chinese patent CN115774062A discloses a method for determining related substances of 3,5-dichlorobenzoic acid. The method uses a Waters Atlantis T3 (4.6mm×250mm, 5μm) chromatographic column and acetonitrile and 0.05%-0.5% phosphoric acid solution as mobile phases for gradient elution, and separates and determines 6 impurities in 3,5-dichlorobenzoic acid. However, when the method is used to determine 9 impurities in 2,5-dichlorobenzoic acid, the impurities are not easy to separate and the chromatographic peaks overlap. Therefore, it is necessary to develop a new method for the determination of related substances of 2,5-dichlorobenzoic acid, to effectively control the quality of the ixazomib citrate raw material and ensure the safety of medication. Summary of the invention

[0013] The invention provides a detection method for separating and determining multiple related substances in 2,5-dichlorobenzoic acid, a starting material of ixazomib citrate. The method is simple to operate, can quickly separate and determine multiple isomers of 2,5-dichlorobenzoic acid and other impurities, and helps to effectively control the quality of the ixazomib citrate raw material.

[0014] According to a method for detecting related substances in 2,5-dichlorobenzoic acid provided by the present invention, the related substances are the following 9 impurities:

[0015]

[0016] The method is characterized in that the detection is carried out by reverse phase high performance liquid chromatography, the chromatographic conditions of the method include a chromatographic column using octadecylsilane bonded silica gel as filler; the mobile phase is a mixed solution consisting of an aqueous phosphoric acid solution and acetonitrile, and gradient elution is carried out by changing the ratio of the aqueous phosphoric acid solution and acetonitrile in the mobile phase; the flow rate of the mobile phase is 0.9 to 1.1 ml / min; and the temperature of the chromatographic column is 20 to 30°C.

[0017] The present invention found in the process of investigating the chromatographic column that the commonly used reversed phase liquid chromatography columns cannot separate all impurities, and there is an overlap of impurity peaks. The chromatographic column used in the present invention is preferably an Inertsil ODS-SP column (250mm×4.6mm, 5μm), which is a reversed phase liquid chromatography column using octadecylsilane bonded silica gel as a filler (C18) and has a low carbon content of 8.5%. Studies have found that only this chromatographic column can separate all impurity peaks. The chromatographic column of the present invention includes a chromatographic column with equivalent performance to the Inertsil ODS-SP column.

[0018] Furthermore, the mobile phase flow rate of the chromatographic conditions of the detection method of the present invention is preferably 1.0 ml / min; and the chromatographic column temperature is preferably 25°C.

[0019] In addition, the detector used in the chromatographic conditions of the present invention is an ultraviolet detector with a detection wavelength of 200 nm to 230 nm, preferably 225 nm.

[0020] The present invention uses an Inertsil ODS-SP column to study and investigate the mobile phase and finds that when the pH of the phosphoric acid solution in the mobile phase is 3.0-3.5, all impurities can be effectively separated. Preferably, the pH of the phosphoric acid solution is 3.0-3.1, with the best separation and the best peak shape.

[0021] The mobile phase composed of phosphoric acid solution and acetonitrile of the present invention is gradient eluted by changing the ratio of phosphoric acid aqueous solution and acetonitrile in the mobile phase. The specific method is to use phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution according to the following table:

[0022]

[0023] During the elution process, the sum of the mobile phase A ratio and the mobile phase B ratio is 100%, wherein the mobile phase A ratio refers to the percentage of mobile phase A in the total volume of the eluent, and the mobile phase B ratio refers to the percentage of mobile phase B in the total volume of the eluent.

[0024] The concentration of the test solution used in the present invention is 0.1 mg / ml to 1.0 mg / ml, preferably 0.5 mg / ml.

[0025] The detection method of the present invention comprises the following steps:

[0026] 1) Preparation of test solution: Take an appropriate amount of 2,5-dichlorobenzoic acid, accurately weigh it, dissolve it in 50% acetonitrile solution and dilute it to make a solution containing about 0.5 mg per 1 ml.

[0027] 2) Preparation of control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with 50% acetonitrile solution, and shake well.

[0028] 3) Sample determination: Accurately measure the test solution and control solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0029] The invention provides a detection method for separating and determining multiple related substances in 2,5-dichlorobenzoic acid, a starting raw material of ixazomib citrate. The method adopts a reversed-phase high performance liquid chromatography method using a C18 liquid chromatography column, adopts a phosphoric acid solution and an acetonitrile mixed solution as a mobile phase, and performs gradient elution by changing the ratio of two solvents in the mobile phase. The method can effectively separate and determine nine impurities such as multiple isomers in 2,5-dichlorobenzoic acid, has good separation degree and peak shape, and can help effectively control the quality of the ixazomib citrate raw material.

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation examples.

[0031] Instruction Manual

[0032] Figure 1 Spectrum of blank solution for specificity test

[0033] Figure 2 Specificity test resolution solution spectrum

[0034] Figure 3 Detection spectrum of related substances in 2,5-dichlorobenzoic acid samples

[0035] Figure 4 Comparative Example Separation Solution Spectrum DETAILED DESCRIPTION

[0036] Example 1 Investigation of separation effects of different chromatographic columns

[0037] The following chromatographic columns with different carbon loadings were used for the test under the following chromatographic conditions.

[0038] Chromatographic column:

[0039] Solution 1 Inertsil ODS-SP (4.6×250 mm, 5 μm), carbon 18 column, carbon loading 8.5% Solution 2 Ecosil C18 (4.6×250 mm, 5 μm), carbon 18 column, carbon loading 9.5% Solution 3 Atlantis T3 (4.6×250 mm, 5 μm), carbon 18 column, carbon loading 14% Solution 4 YMC-Pack ODS-AM (4.6×250 mm, 5 μm), carbon 18 column, carbon loading 17%

[0040] Chromatographic conditions: pH 3.0 phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution according to the table below; flow rate 1.0 ml per minute; column temperature 25°C; detection wavelength 225 nm; injection volume 15 μl.

[0041]

[0042]

[0043] Impurity localization solutions: Take appropriate amounts of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8 and impurity A9, weigh accurately, add 50% acetonitrile to dissolve and quantitatively dilute to make solutions containing about 0.5 mg per 1 ml.

[0044] Separation solution: Accurately weigh an appropriate amount of 2,5-dichlorobenzoic acid, add an appropriate amount of each positioning solution of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8, and impurity A9, add 50% acetonitrile solution to dissolve and dilute to make a mixed solution containing 0.5 mg of 2,5-dichlorobenzoic acid and 1 μg of each of other impurities in each ml.

[0045] Detection steps: Accurately measure 15 μl of the separation solution and inject it into the liquid chromatograph, and record the chromatogram.

[0046] The results showed that the separation degree between impurities in Scheme 1 was greater than 1.5, and there were two impurity chromatographic peaks overlapping in Schemes 3 and 4. The minimum separation degree of Scheme 1 was 1.74, and the minimum separation degree of Scheme 2 was 1.40. Scheme 1 had the best separation effect.

[0047] Example 2 Investigation of Separation Effects of Different Mobile Phases

[0048] Inertsil ODS-SP (4.6×250 mm, 5 μm) was used as the chromatographic column and the following different mobile phases were used for the experiments.

[0049] Mobile Phase:

[0050] Solution 1 Mobile phase A: pH 3.0 phosphoric acid aqueous solution; Mobile phase B: acetonitrile Solution 5 Mobile phase A: 0.01% trifluoroacetic acid solution; mobile phase B: acetonitrile. Solution 6 Mobile phase A: 25 mmol / L potassium dihydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid); mobile phase B: acetonitrile.

[0051] Perform gradient elution according to the table below; flow rate is 1.0 ml per minute; column temperature is 25°C; detection wavelength is 225 nm; injection volume is 15 μl.

[0052]

[0053] Impurity localization solutions: Take appropriate amounts of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8 and impurity A9, weigh accurately, add 50% acetonitrile to dissolve and quantitatively dilute to make solutions containing about 0.5 mg per 1 ml.

[0054] Separation solution: Accurately weigh an appropriate amount of 2,5-dichlorobenzoic acid, add an appropriate amount of each positioning solution of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8, and impurity A9, add 50% acetonitrile solution to dissolve and dilute to make a mixed solution containing 0.5 mg of 2,5-dichlorobenzoic acid and 1 μg of each of other impurities in each ml.

[0055] Detection steps: Accurately measure 15 μl of the separation solution and inject it into the liquid chromatograph, and record the chromatogram.

[0056] The results showed that when 0.01% trifluoroacetic acid solution and acetonitrile were used as the mobile phase, there was overlap of impurity chromatographic peaks; when 25 mmol / L potassium dihydrogen phosphate solution (phosphoric acid adjusted to pH 3.5) and acetonitrile were used as the mobile phase, the minimum separation degree of adjacent impurities was 1.28, and the separation effect was worse than that of scheme 1 using pH 3.0 phosphoric acid aqueous solution and acetonitrile as the mobile phase.

[0057] Example 3 Investigation of the mobile phase separation effect of phosphoric acid solutions at different pH values

[0058] The experiment was conducted using Inertsil ODS-SP (4.6×250mm, 5μm) as the chromatographic column and the following mobile phases consisting of phosphoric acid solutions with different pH values ​​and acetonitrile:

[0059] Solution 7 Mobile phase A: 0.1% phosphoric acid solution (pH 2.2); mobile phase B: acetonitrile. Solution 8 Mobile phase A: phosphoric acid solution (pH 2.7); mobile phase B: acetonitrile. Solution 9 Mobile phase A: phosphoric acid solution (pH 2.9); mobile phase B: acetonitrile. Solution 1 Mobile phase A: phosphoric acid solution (pH 3.0); mobile phase B: acetonitrile. Solution 10 Mobile phase A: phosphoric acid solution (pH 3.1); mobile phase B: acetonitrile. Solution 11 Mobile phase A: phosphoric acid solution (pH 3.2); mobile phase B: acetonitrile. Solution 12 Mobile phase A: phosphoric acid solution (pH 3.3); mobile phase B: acetonitrile. Solution 13 Mobile phase A: phosphoric acid solution (pH 3.4); mobile phase B: acetonitrile. Solution 14 Mobile phase A: phosphoric acid solution (pH 3.5); mobile phase B: acetonitrile.

[0060] The method for preparing the phosphoric acid solution is as follows: take 1000 ml of water, add dilute phosphoric acid therein, stir while adding, and stop adding when the pH value reaches the required pH value.

[0061] Use the mobile phase in the above table for gradient elution as shown below; the flow rate is 1.0 ml per minute; the column temperature is 25°C; the detection wavelength is 225 nm; and the injection volume is 15 μl.

[0062]

[0063]

[0064] Impurity localization solutions: Take appropriate amounts of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8 and impurity A9, weigh accurately, add 50% acetonitrile to dissolve and quantitatively dilute to make solutions containing about 0.5 mg per 1 ml.

[0065] Separation solution: Accurately weigh an appropriate amount of 2,5-dichlorobenzoic acid, add an appropriate amount of each positioning solution of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8, and impurity A9, add 50% acetonitrile solution to dissolve and dilute to make a mixed solution containing about 0.5 mg of 2,5-dichlorobenzoic acid and about 1 μg of each of other impurities in each ml.

[0066] Detection steps: Accurately measure 15 μl of the separation solution and inject it into the liquid chromatograph, and record the chromatogram.

[0067] The test results of Scheme 1 and Schemes 7 to 14 are as follows:

[0068] Mobile phase Test results (minimum separation of adjacent impurities) Mobile phase A: 0.1% phosphoric acid solution (pH 2.2); mobile phase B: acetonitrile. Impurity chromatographic peaks overlap and cannot be effectively separated Mobile phase A: phosphoric acid solution (pH 2.7); mobile phase B: acetonitrile. Impurity chromatographic peaks overlap and cannot be effectively separated Mobile phase A: phosphoric acid solution (pH 2.9); mobile phase B: acetonitrile. Impurity chromatographic peaks overlap and cannot be effectively separated Mobile phase A: phosphoric acid solution (pH 3.0); mobile phase B: acetonitrile. 1.97 Mobile phase A: phosphoric acid solution (pH 3.1); mobile phase B: acetonitrile. 3.08 Mobile phase A: phosphoric acid solution (pH 3.2); mobile phase B: acetonitrile. 1.58 Mobile phase A: phosphoric acid solution (pH 3.3); mobile phase B: acetonitrile. 1.54 Mobile phase A: phosphoric acid solution (pH 3.4); mobile phase B: acetonitrile. 1.51 Mobile phase A: phosphoric acid solution (pH 3.5); mobile phase B: acetonitrile. 1.52

[0069] From the above test results, it can be seen that when 0.1% phosphoric acid solution (pH 2.2) is used, there is overlap of impurity chromatographic peaks. When the phosphoric acid solution has a pH of 3.0-3.5, the minimum separation degree of adjacent impurities is greater than 1.5, which meets the requirements. The separation efficiency is optimal at pH 3.0 and 3.1.

[0070] Example 4: Verification test of the method of the present invention

[0071] Chromatographic conditions: Inertsil ODS-SP (4.6×250mm, 5μm) as the chromatographic column; phosphoric acid solution (pH3.0) as mobile phase A, acetonitrile as mobile phase B, gradient elution according to the table below; flow rate 1.0ml per minute; column temperature 25°C; detection wavelength 225nm; injection volume 15μl.

[0072]

[0073] Test solution: Take an appropriate amount of 2,5-dichlorobenzoic acid, weigh it accurately, dissolve it in 50% acetonitrile solution and dilute it to make a solution containing about 0.5 mg per 1 ml.

[0074] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with 50% acetonitrile solution, and shake well.

[0075] Impurity localization solutions: Take appropriate amounts of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8 and impurity A9, weigh accurately, add 50% acetonitrile to dissolve and quantitatively dilute to make solutions containing about 0.5 mg per 1 ml.

[0076] Separation solution: Accurately weigh an appropriate amount of 2,5-dichlorobenzoic acid, add an appropriate amount of each positioning solution of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8, and impurity A9, add 50% acetonitrile solution to dissolve and dilute to make a mixed solution containing about 0.5 mg of 2,5-dichlorobenzoic acid and about 1 μg of each of other impurities in each ml.

[0077] (1) Specificity test

[0078] Take blank solvent, test solution, impurity location solution, and resolution solution and inject them separately to investigate the interference of blank solution on the determination and the separation of impurities. The experimental results show that blank solvent has no interference, and the resolution between each impurity is greater than 1.5, which meets the determination requirements (blank solvent and resolution solution spectrum see attached Figure 1-2 ).

[0079] Specificity test results

[0080]

[0081]

[0082] (2) Limit of detection and limit of quantification

[0083] Each impurity location solution was diluted step by step to the test sample solution concentration of 0.006% and 0.01%, respectively, as the detection limit and quantification limit of each impurity, the signal-to-noise ratio of each impurity peak in the chromatogram of the quantification limit solution was greater than 10, and the signal-to-noise ratio of each impurity peak in the chromatogram of the detection limit solution was greater than 3, indicating that the detection limit and quantification limit of this method meet the determination requirements.

[0084]

[0085] (3) Linearity and range

[0086] Accurately measure 5 ml of the positioning solutions of impurity A1, impurity A2, impurity A3, impurity A4 and impurity A5, respectively, place them in the same 50 ml volumetric flask, dilute to the scale with solvent, and shake well to obtain mixed stock solution 1; accurately measure 5 ml of the positioning solutions of impurity A6, impurity A7, impurity A8, impurity A9 and 2,5-dichlorobenzoic acid, respectively, place them in the same 50 ml volumetric flask, dilute to the scale with solvent, and shake well to obtain mixed stock solution 2.

[0087] Linear solution 1: Accurately measure 10 ml of each mixed stock solution 1 and mixed stock solution 2, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain linear solution 6 (1%); accurately measure 5 ml of linear solution 6, place it in a 50 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain linear solution 3 (0.1%); accurately measure 5 ml of linear solution 3, place it in a 25 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain linear solution 1 (0.02%).

[0088] Linear solution 2: Accurately measure 5 ml of linear solution 6, place it in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain linear solution 2 (0.05%).

[0089] Linear solution 4: Accurately measure 5 ml of linear solution 6, place it in a 25 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain linear solution 4 (0.2%).

[0090] Linear solution 5: Accurately measure 5 ml of mixed stock solution 1 and mixed stock solution 2, place them in the same 25 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain linear solution 7 (2%); Accurately measure 5 ml of linear solution 7, place it in a 20 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain linear solution 5 (0.5%).

[0091] Accurately measure linear solution 1, linear solution 2, linear solution 3, linear solution 4, linear solution 5, linear solution 6, and linear solution 7, and inject them into the liquid chromatograph respectively to record the chromatogram. Perform linear regression on the concentration of the corresponding solution based on the peak area of ​​each target peak, examine its linear correlation coefficient, and calculate the correction factor of each impurity.

[0092] The peak areas of the main components and known impurities were well correlated with the concentrations when the concentrations were between 0.02% and 2.0% of the test solution, and the correlation coefficients were between 0.9999 and 1.0000.

[0093] project Concentration (equivalent to the percentage of the test product) Linear equation / correlation coefficient r Correction Factor principal component 0.11~10.93μg / ml (0.02%~2.0%) y=50017x-753.18; r=1.0000 NA Impurity A1 0.11~11.19μg / ml (0.02%~2.0%) y=40223X-1240.1; r=1.0000 1.24 Impurity A2 0.10~10.34μg / ml (0.02%~2.0%) y=32683x-470.03; r=1.0000 1.53 Impurity A3 0.11~10.65μg / ml (0.02%~2.0%) y=36745x-386.29; r=1.0000 1.36 Impurity A4 0.10~10.17μg / ml (0.02%~2.0%) y=39010x-973.82; r=1.0000 1.28 Impurity A5 0.11~10.51μg / ml (0.02%~2.0%) y=37106x-208.23; r=1.0000 1.35 Impurity A6 0.11~10.56μg / ml (0.02%~2.0%) y=53389x-446.19; r=1.0000 0.94 Impurity A7 0.10~10.29μg / ml (0.02%~2.0%) y=62915x-607.71; r=1.0000 0.79 Impurity A8 0.11~10.86μg / ml (0.02%~2.0%) y=51042x-466.55; r=1.0000 0.98 Impurity A9 0.12~12.50μg / ml (0.02%~2.0%) y=53412x+123.14; r=0.9999 0.94

[0094] (4) Accuracy test

[0095] Mixed impurity stock solution: Accurately measure 5 ml of each impurity location solution into the same 50 ml volumetric flask, dilute to the scale with 50% acetonitrile solution, and shake well.

[0096] 0.05% mixed impurity reference solution: Accurately measure 1 ml of the mixed impurity stock solution, place it in a 200 ml volumetric flask, dilute to the scale with 50% acetonitrile solution, and shake well.

[0097] 0.1% mixed impurity reference solution: Accurately measure 1 ml of the mixed impurity stock solution, place it in a 100 ml volumetric flask, dilute to the scale with 50% acetonitrile solution, and shake well.

[0098] 0.5% mixed impurity reference solution: Accurately measure 5 ml of the mixed impurity stock solution, place it in a 100 ml volumetric flask, dilute to the scale with 50% acetonitrile solution, and shake well.

[0099] 1.0% mixed impurity reference solution: Accurately measure 10 ml of the mixed impurity stock solution, place it in a 100 ml volumetric flask, dilute to the scale with 50% acetonitrile solution, and shake well.

[0100] 0.05% limit recovery solution: Accurately weigh about 10 mg of 2,5-dichlorobenzoic acid sample, place in a 20 ml volumetric flask, add 0.05% mixed impurity reference solution to dissolve and dilute to scale, shake well. Prepare 3 copies in parallel.

[0101] 0.1% limit recovery solution: Accurately weigh about 10 mg of 2,5-dichlorobenzoic acid sample, place in a 20 ml volumetric flask, add 0.1% mixed impurity reference solution to dissolve and dilute to scale, shake well. Prepare 3 copies in parallel.

[0102] 0.5% limit recovery solution: Accurately weigh about 10 mg of 2,5-dichlorobenzoic acid sample, place in a 20 ml volumetric flask, add 0.5% mixed impurity reference solution to dissolve and dilute to scale, shake well. Prepare 3 copies in parallel.

[0103] 1.0% limit recovery solution: Accurately weigh about 10 mg of 2,5-dichlorobenzoic acid sample, place in a 20 ml volumetric flask, add 1.0% mixed impurity reference solution to dissolve and dilute to scale, shake well. Prepare 3 copies in parallel.

[0104] Control solution: Accurately take 1 ml of each recovery solution mentioned above, place it in a 100 ml volumetric flask, dilute to the scale with 50% acetonitrile solution, and shake well.

[0105] Accurately measure each recovery solution and its corresponding reference solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the content of each impurity in the recovery solution by the principal component self-reference method with correction factor added.

[0106] The average recovery rates of each known impurity were between 80% and 120%, and the RSD values ​​of the 12 recoveries were all less than 10%, indicating that the method had good accuracy.

[0107]

[0108]

[0109] (6) Repeatability test

[0110] Test solution: Take an appropriate amount of 2,5-dichlorobenzoic acid, dissolve it in 50% acetonitrile solution and dilute it to make a solution containing about 0.5 mg per 1 ml. Prepare 6 copies in parallel.

[0111] Control solution: Accurately measure an appropriate amount of the test sample solution and quantitatively dilute it with 50% acetonitrile solution to make a solution containing approximately 5 μg per 1 ml.

[0112] Accurately measure the blank solution, test solution and reference solution and inject them into the liquid chromatograph respectively, record the chromatogram and calculate the content of each impurity by the main component self-reference method with correction factor.

[0113] The measurement was repeated 6 times, and the measured values ​​were close, with a difference of less than 0.1%, indicating that this method has good repeatability.

[0114]

[0115] Example 5 Detection of Related Substances in 2,5-Dichlorobenzoic Acid Samples

[0116] Chromatographic conditions:

[0117] Chromatographic column: Inertsil ODS-SP (4.6×250 mm, 5 μm); column temperature: 25°C;

[0118] Detection wavelength: 225nm (UV detector);

[0119] Injection volume: 15 μl;

[0120] Mobile phase: phosphoric acid solution (pH 3.0) as mobile phase A, acetonitrile as mobile phase B; flow rate: 1.0 ml; gradient elution according to the following table:

[0121]

[0122]

[0123] Sample solution: Take an appropriate amount of 2,5-dichlorobenzoic acid sample, weigh it accurately, add 50% acetonitrile solution to dissolve it and dilute it to make a solution containing about 0.5 mg per 1 ml.

[0124] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with 50% acetonitrile solution, and shake well.

[0125] Sample determination: Accurately measure the sample solution and the control solution and inject them into the liquid chromatograph respectively, and record the chromatogram (add the attached Figure 3 ).

[0126] Measurement results: impurity A5 content 0.02%, impurity A6 content 0.01%, impurity A7 content 0.01%, other impurities were not detected.

[0127] Comparative Example The patent (publication number: CN115774062A) method is used to separate and determine the related substances in 2,5-dichlorobenzoic acid. Chromatographic conditions:

[0128] Chromatographic column: Waters Atlantis T3 (4.6 mm × 250 mm, 5 μm); column temperature: 35 °C;

[0129] Detection wavelength: 210nm (UV detector);

[0130] Injection volume: 10 μl;

[0131] Mobile phase: 0.1% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B; flow rate: 1.0 ml; gradient elution according to the following table:

[0132]

[0133] Impurity localization solutions: Take appropriate amounts of impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, impurity A8 and impurity A9, weigh accurately, add 50% acetonitrile to dissolve and quantitatively dilute to make solutions containing about 0.5 mg per 1 ml.

[0134] Separation solution: Accurately weigh an appropriate amount of 2,5-dichlorobenzoic acid, add an appropriate amount of 2,5-dichlorotoluene, impurity A1, impurity A2, impurity A3, impurity A4, impurity A5, impurity A6, impurity A7, and impurity A8 positioning solutions, add 50% acetonitrile solution to dissolve and dilute to make a mixed solution containing about 0.5 mg of 2,5-dichlorobenzoic acid and about 1 μg of each of the other impurities per 1 ml.

[0135] Detection steps: Accurately measure 10 μl of the separation solution and inject it into the liquid chromatograph, and record the chromatogram. The chromatogram is attached. Figure 4 .

[0136] Depend on Figure 4 It can be seen that there is chromatographic peak overlap at the retention time of 17.673 minutes, and this method fails to effectively separate the 2,5-dichlorobenzoic acid impurity.

Claims

1. A method for detecting related substances in 2,5-dichlorobenzoic acid, wherein the related substances are the following 9 impurities: It is characterized in that Reverse phase high performance liquid chromatography is used for detection. The chromatographic conditions of the method include using octadecylsilane bonded silica gel as a chromatographic column as a filler; the mobile phase is a mixed solution consisting of phosphoric acid aqueous solution and acetonitrile, and gradient elution is performed by changing the ratio of phosphoric acid aqueous solution and acetonitrile in the mobile phase; the flow rate of the mobile phase is 0.9-1.1 ml / min; and the temperature of the chromatographic column is 20-30°C.

2. The method according to claim 1, characterized in that The model of the chromatographic column is Inertsil ODS-SP column.

3. The method according to claim 1, characterized in that The pH of the phosphoric acid aqueous solution is 3.0-3.

5.

4. The method according to claim 3, characterized in that The pH of the phosphoric acid aqueous solution is 3.0-3.

1.

5. The method according to claim 1, characterized in that The specific method of the gradient elution is to use phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and elute according to the following gradient:

6. The method according to claim 1, wherein the concentration of the test solution is 0.5 mg / ml.

7. The method according to claim 1, wherein the flow rate of the mobile phase is 1.0 ml / min; and the temperature of the chromatographic column is 25°C.

8. The method according to claim 1, wherein the detection method comprises the following steps: 1) Preparation of test solution: Take an appropriate amount of 2,5-dichlorobenzoic acid, accurately weigh it, dissolve it in 50% acetonitrile solution and dilute it to make a solution containing about 0.5 mg per 1 ml. 2) Preparation of control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with 50% acetonitrile solution, and shake well. 3) Sample determination: Accurately measure the test solution and control solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

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