Analysis method of related substances of 4-chloro-3-trifluoromethyl phenyl isocyanate

Abstract

The invention belongs to the technical field of chemical analysis. Relates to a method for detecting BP007-SM3 related substances, in particular to a method for detecting BP007-SM3 related substances {4-chloro-3-(trifluoromethyl) aniline, 1, 3-bis [4-chloro-3-(trifluoromethyl) phenyl] urea, 2-chloro-5-nitro-trifluoromethyl benzene, methyl [(3-trifluoromethyl) phenyl] carbamate, methyl [2-chloro-3-(trifluoromethyl) phenyl] carbamate and 2-chloro-3-(trifluoromethyl) aniline}. Particularly relates to a chromatographic column taking phenylsilane bonded silica gel as a filling agent, which adopts a diode array detector or an ultraviolet detector, takes a mixed solution of a phosphate buffer solution, acetonitrile and methanol as an eluent, and performs elution according to a gradient mode. And a detection method which is simple and convenient to operate, high in specificity, high in accuracy and high in sensitivity is provided for detecting related substances of BP007-SM3.

Description

Field of the Invention

[0001] The invention belongs to the technical field of drug analysis and provides a method for detecting related substances in 4-chloro-3-trifluoromethylphenyl isocyanate. Background of the Invention

[0002] 4-Chloro-3-trifluoromethylphenyl isocyanate (hereinafter referred to as BP007-SM3) is an important starting material for the synthesis of regorafenib raw materials. The impurities in this product will be introduced into the drug in the form of prototype or conductive impurities in the subsequent synthesis process, which will eventually affect the quality of the drug and further affect the safety of drug use. Therefore, it is necessary to fully study the relevant substances in this product to ensure that the quality is controllable. The structural formula of BP007-SM3 is as follows:

[0003]

[0004] CN113466395 discloses a method for detecting BP007-SM3 using a high performance liquid chromatography-mass spectrometer, but no further determination and research on BP007-SM3 related impurities is conducted. Currently, there is an urgent need for an analytical method for BP007-SM3 and related substances with strong specificity, good precision, high accuracy, excellent separation effect, and good durability to provide effective support for its production process and quality control. Summary of the invention

[0005] The present invention provides a method for detecting related substances of BP007-SM3, which is characterized by adopting a high performance liquid chromatography method, using a reverse phase chromatography column, and eluting in an isocratic or gradient manner.

[0006] In some typical embodiments, mobile phase A and / or mobile phase B are used as eluents, wherein mobile phase A is phosphate buffer [10 mmol / L potassium dihydrogen phosphate (pH adjusted to 2.4 with phosphoric acid)]-acetonitrile (95:5); mobile phase B is acetonitrile-methanol (80:20); in some embodiments, the concentration of the phosphate buffer is 8 mmol / L to 12 mmol / L; preferably, the concentration of the phosphate buffer is 10 mmol / L.

[0007] In some embodiments, the phosphate buffer needs to have a pH value of 2.3 to 2.5 under the phosphoric acid condition. Preferably, the pH value of the phosphate buffer is 2.4.

[0008] In some embodiments, the phosphate buffer-acetonitrile ratio is (93-97):(7-3), preferably 95:5.

[0009] In some embodiments, the reverse phase chromatography column uses non-polar stationary as filler; preferably, the reverse phase chromatography uses phenylsilane bonded silica gel as filler; more preferably, the reverse phase chromatography column is Kromasil 100-5phenyl, with a specification of 4.6mm×250mm, 5μm.

[0010] In some embodiments, the column temperature is 28°C to 32°C; preferably, the column temperature is 30°C.

[0011] In some embodiments, the flow rate of the eluent is 0.7-0.9 ml / min; preferably, the flow rate of the eluent is 0.8 ml / min.

[0012] In some embodiments, the detection method is performed on a high performance liquid chromatograph using a diode array detector or an ultraviolet detector; preferably, the analysis method is performed on a high performance liquid chromatograph using an ultraviolet detector, and its detection wavelength is 246 nm.

[0013] In some embodiments, the related substances in BP007-SM3 include impurity BP700-SM3-A [chemical name: 4-chloro-3-(trifluoromethyl)aniline], whose structural formula is as follows:

[0014]

[0015] In some embodiments, the related substances in BP007-SM3 include impurity BP700-SM3-B {chemical name: 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]urea}, whose structural formula is as follows:

[0016]

[0017] In some embodiments, the related substances in BP007-SM3 include impurity BP700-SM3-E (chemical name: 2-chloro-5-nitro-trifluoromethylbenzene), whose structural formula is as follows:

[0018]

[0019] In some embodiments, the related substances in BP007-SM3 include the impurity BP700-SM3-F {chemical name: methyl [(3-trifluoromethyl) phenyl] carbamate}, whose structural formula is as follows:

[0020]

[0021] In some embodiments, the related substances in BP007-SM3 include impurity BP700-SM3-H [chemical name: 2-chloro-3-(trifluoromethyl)aniline], whose structural formula is as follows:

[0022]

[0023] In some embodiments, the related substances in BP007-SM3 include impurity BP700-SM3-I {chemical name: methyl [2-chloro-3-(trifluoromethyl)phenyl] carbamate}, whose structural formula is as follows:

[0024]

[0025] On the other hand, the present invention provides a method for detecting BP007-SM3 related substances, characterized in that: the method is a high performance liquid chromatography method, the method uses a reverse phase chromatographic column, the detector is a UV absorption detector, the detection wavelength is 246nm; flow rate: 0.8ml / min; column temperature: 30°C; injection volume: 5μl; injector temperature: 4°C; mobile phase A: phosphate buffer [10mmol / L potassium dihydrogen phosphate (phosphoric acid adjusted to pH 2.4)]-acetonitrile (95:5), mobile phase B: acetonitrile-methanol (80:20); gradient elution is performed according to the following table:

[0026]

[0027] In some typical embodiments, the present invention provides a method for detecting related substances of BP007-SM3, characterized by the following steps:

[0028] (1) Test solution: Take an appropriate amount of the product, dissolve it in anhydrous methanol and dilute it to make a solution containing about 5 mg per 1 ml as the test sample stock solution; accurately measure an appropriate amount and dilute it with acetonitrile-water (70:30) to make a solution containing about 0.5 mg per 1 ml.

[0029] (2) Control solution: Accurately measure an appropriate amount of the test sample solution and dilute it with acetonitrile-water (70:30) to make a solution containing approximately 5 μg per 1 ml.

[0030] (3) System suitability solution: Take an appropriate amount of impurity BP007-SM3-B reference substance, dissolve it in acetonitrile and dilute it to make a solution containing about 0.5 mg per 1 ml; take an appropriate amount of impurity BP007-SM3-A reference substance, impurity BP007-SM3-E reference substance, impurity BP007-SM3-F reference substance (methanol-derivative impurity of impurity BP007-SM3-C), impurity BP007-SM3-H reference substance, impurity BP007-SM3-I reference substance (methanol-derivative impurity of impurity BP007-SM3-D), dissolve it in acetonitrile-water (70:30) and dilute it to make a solution containing about 0.5 mg per 1 ml as the stock solutions of each impurity; take an appropriate amount of the test sample stock solution and each impurity stock solution, place them in the same volumetric flask, and dilute them with acetonitrile-water (70:30) to make a solution containing about 0.5 mg of BP007-SM3 and 5 μg of each impurity per 1 ml.

[0031] (4) Calculate the related substances in BP007-SM3 according to the self-control method.

[0032] It is easy for those skilled in the art to understand that some of the operating steps of the analytical method described in this application can be replaced without affecting the results. For example, the preparation steps of the test solution, the control solution, and the system suitability solution can be replaced at will.

[0033] In this application, related substances are also expressed as impurities.

[0034] In the present application, an appropriate amount means that the amount of each compound is within the detection line or quantitative limit of its high performance liquid chromatography according to the experimental purpose.

[0035] In the present application, suitable concentration means that the concentration of each compound is within the detection limit or quantitative limit of its high performance liquid chromatography according to the experimental purpose.

[0036] Unless otherwise specified, the abbreviations in this application have the following meanings:

[0037] min: minute;

[0038] ml: milliliter;

[0039] μl: microliter;

[0040] mmol / L: millimole per liter;

[0041] LOQ: limit of quantitation;

[0042] LOD: limit of detection;

[0043] RSD: relative standard deviation.

[0044] The method for detecting related substances in BP007-SM3 provided by the present invention can effectively separate BP007-SM3, impurity BP700-SM3-A, impurity BP700-SM3-B, impurity BP700-SM3-E, impurity BP700-SM3-F, impurity BP700-SM3-H and impurity BP700-SM3-I by selecting a suitable mobile phase and controlling the concentration and flow rate of the mobile phase, the chromatographic column, the column temperature and other conditions. The method has good specificity, good accuracy, good reproducibility and good durability, and can qualitatively and quantitatively detect multiple impurities in BP007-SM3. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 System suitability chromatogram of Example 2 DETAILED DESCRIPTION

[0046] Unless otherwise specified, the reagents and raw materials used in the following examples of the present invention are all commercially available.

[0047] Example 1 Determination of the test method

[0048] Chromatographic column: Phenylsilane bonded silica gel chromatographic column (Kromasil 100-5Phenyl, 4.6mm×250mm, 5μm or chromatographic column with equivalent performance)

[0049] Detection wavelength: 246nm;

[0050] Flow rate: 0.8ml / min;

[0051] Column temperature: 30°C;

[0052] Injection volume: 5 μl;

[0053] Injector temperature: 4 °C;

[0054] Mobile phase A: phosphate buffer [10 mmol / L potassium dihydrogen phosphate (pH adjusted to 2.4 with phosphoric acid)]-acetonitrile (95:5);

[0055] Mobile phase B: acetonitrile-methanol (80:20);

[0056] Perform gradient elution according to the following table:

[0057]

[0058]

[0059] Sample preparation:

[0060] (1) Test solution: Take an appropriate amount of the product, dissolve it in anhydrous methanol and dilute it to a solution containing about 5 mg per 1 ml as the test stock solution; accurately measure an appropriate amount and dilute it with acetonitrile-water (70:30) to a solution containing about 10 mg per 1 ml.

[0061] 0.5mg of solution.

[0062] (2) Control solution: Accurately measure an appropriate amount of the test solution and dilute it with acetonitrile-water (70:30) to make a solution containing approximately

[0063] 5μg of solution.

[0064] (3) System suitability solution: Take an appropriate amount of impurity BP007-SM3-B reference substance, dissolve it in acetonitrile and dilute it to make a solution containing about 0.5 mg per 1 ml, take impurity BP007-SM3-A reference substance, impurity BP007-SM3-E reference substance, impurity BP007-SM3-F reference substance (methanol-derivatized impurity of impurity BP007-SM3-C), impurity BP007-SM3-

[0065] H reference substance, impurity BP007-SM3-I reference substance (methanol-derivatized impurity of impurity BP007-SM3-D)

[0066] Take an appropriate amount of the test sample stock solution and each impurity stock solution, place them in the same volumetric flask, and dilute them with acetonitrile-water (70:30) to prepare a solution containing approximately 0.5 mg of BP007-SM3 and 5 μg of each impurity per 1 ml.

[0067] Example 2 Specificity Test

[0068] Solvent: acetonitrile-water-(70:30)

[0069] Impurity stock solution: Take about 10 mg of impurity BP007-SM3-A, impurity BP007-SM3-F, impurity BP007-SM3-I, impurity BP007-SM3-E, and impurity BP007-SM3-H respectively, weigh accurately, dissolve in solvent and dilute to make a solution containing about 0.5 mg per 1 ml, take about 10 mg of impurity BP007-SM3-B, dissolve in acetonitrile and dilute to make a solution containing about 0.5 mg per 1 ml, as the stock solution of each impurity.

[0070] Impurity localization solution: Take 1 ml of each impurity stock solution, place it in different 10 ml volumetric bottles, dilute to the scale with solvent, and shake well.

[0071] BP007-SM3 reference substance stock solution: Take an appropriate amount of BP007-SM3 reference substance, weigh accurately, dissolve in solvent and dilute to make a solution containing approximately 0.5 mg per 1 ml.

[0072] Test sample solution: Take about 100 mg of this product, weigh it accurately, dissolve it in anhydrous methanol and dilute it to make a solution containing about 5 mg per 1 ml, which is used as the test sample stock solution; accurately measure an appropriate amount, dissolve it in solvent and dilute it to make a solution containing about 0.5 mg per 1 ml.

[0073] Preparation of system suitability solution: Take about 50 mg of BP007-SM3 reference substance, accurately weigh it, add anhydrous methanol to dissolve and dilute it to make a solution containing about 5 mg per 1 ml as the stock solution, accurately measure 5 ml of the stock solution, put it in a 50 ml volumetric flask, accurately measure impurities BP007-SM3-A, impurity BP007-SM3-B, impurity BP007-SM3-F, impurity BP007-SM3-I, impurity BP007-SM3-E, impurity B 0.5 ml of P007-SM3-H stock solution was placed in the same 50 ml volumetric flask and diluted with solvent to make a solution containing about 0.5 mg of BP007-SM3 and about 5 μg of impurities BP007-SM3-A, BP007-SM3-B, BP007-SM3-F, BP007-SM3-I, BP007-SM3-E and BP007-SM3-H per 1 ml, which was used as the system suitability solution.

[0074] Take 5 μl of each of the blank solvent, impurity location solution, test solution and system suitability solution, inject them into the liquid chromatograph and record the chromatogram. The experimental results are shown in Table 1.

[0075] Table 1 Specificity results

[0076]

[0077] The results showed that there was no interference in the blank, the separation between the peaks was greater than 1.2, and the system had good applicability.

[0078] Example 3 Limit of Quantitation and Limit of Detection

[0079] Accurately measure appropriate amounts of BP007-SM3 and stock solutions of related substances under the specificity item, prepare a series of solutions, inject into the liquid chromatograph, record the chromatogram, and when the signal-to-noise ratio is ≥9, use it as the quantitative limit solution, and prepare 6 portions in parallel; when the signal-to-noise ratio is ≥3, use it as the detection limit solution. The experimental results are shown in Tables 2 and 3.

[0080] Table 2 Quantitation limit results

[0081]

[0082]

[0083] Table 3 Detection limit results

[0084]

[0085]

[0086] From the above test results, it can be seen that the RSD of the peak area of ​​each impurity quantitative limit is ≤10.0%, and the RSD of the retention time is ≤1.0%.

[0087] Both LOQ and LOD were less than 0.05% of the test sample concentration, and the sensitivity of each impurity met the requirements.

[0088] Example 4 Linearity and Range

[0089] Linear stock solution: Accurately measure 1 ml of impurity BP007-SM3-A, impurity BP007-SM3-F, impurity BP007-SM3-I, impurity BP007-SM3-E, impurity BP007-SM3-H, and BP007-SM3 reference substance stock solution and 5 ml of impurity BP007-SM3-B reference substance stock solution under the specificity item, place them in a 50 ml volumetric flask, and dilute with solvent to make a solution containing approximately 10 μg of BP007-SM3, impurity BP007-SM3-A, impurity BP007-SM3-F, impurity BP007-SM3-I, impurity BP007-SM3-E, and impurity BP007-SM3-H, and 50 μg of impurity BP007-SM3-B per 1 ml.

[0090] Pipette 0.5ml, 1ml, 1ml, 3ml and 2ml of the stock solution respectively, place into 20ml, 25ml, 20ml, 50ml and 20ml volumetric flasks, dilute to the scale with solvent and shake well.

[0091] The linear series solutions and the quantitative limit solution were accurately measured and injected for analysis. The peak area was used to perform linear regression on the concentration. The results are shown in Tables 4 to 10.

[0092] Table 4 BP007-SM3 linearity and range test results

[0093]

[0094]

[0095] Table 5 Impurity BP007-SM3-A linearity and range test results

[0096]

[0097] Table 6 Impurity BP007-SM3-B linearity and range test results

[0098]

[0099]

[0100] Table 7 Impurity BP007-SM3-E linearity and range test results

[0101]

[0102] Table 8 Impurity BP007-SM3-F linearity and range test results

[0103]

[0104] Table 9 Impurity BP007-SM3-I linearity and range test results

[0105]

[0106]

[0107] Table 10 Impurity BP007-SM3-H linearity and range test results

[0108]

[0109] Example 5 Accuracy Test

[0110] Reference substance stock solution: Take about 10 mg of impurity BP007-SM3-A, impurity BP007-SM3-B, impurity BP007-SM3-E, impurity BP007-SM3-H, impurity BP007-SM3-F, and impurity BP007-SM3-I respectively, accurately weigh, place in different 20 ml volumetric bottles, dissolve and dilute to the scale with solvent, shake well, as the respective impurity stock solutions; accurately measure impurity BP007-SM3-A, impurity BP007-SM3-F, impurity BP007-SM3-H, impurity BP007-SM3-F, and impurity BP007-SM3-I respectively. 1 ml of impurity stock solution of M3-I, impurity BP007-SM3-E, impurity BP007-SM3-H, and 5 ml of impurity stock solution of impurity BP007-SM3-B are placed in the same 50 ml volumetric flask and diluted with solvent to make a solution containing about 10 μg of impurity BP007-SM3-A, impurity BP007-SM3-F, impurity BP007-SM3-I, impurity BP007-SM3-E, impurity BP007-SM3-H, and about 50 μg of impurity BP007-SM3-B per 1 ml. Prepare 3 portions in parallel.

[0111] Test sample solution: Take an appropriate amount of the product, dissolve and dilute with anhydrous methanol to make a solution containing about 5 mg of the product per 1 ml. Accurately measure an appropriate amount of the test sample stock solution and dilute with solvent to make a solution containing about 0.5 mg per 1 ml as the test sample solution.

[0112] Reference substance solution: Accurately measure 1 ml of reference substance stock solution, place in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0113] Preparation of recovery test solution: Take an appropriate amount of the product, weigh it accurately, dissolve and dilute it in anhydrous methanol to make a solution containing about 5 mg per 1 ml, accurately measure 2 ml, place it in a 20 ml volumetric flask, add 0.5 ml, 1 ml, and 1.5 ml of the reference stock solution respectively, and dilute it with solvent to make solutions equivalent to 50%, 100%, and 150% of the impurity limit concentration, 3 copies for each concentration.

[0114] Take blank solution, reference solution, test solution and recovery test solution and inject them into liquid chromatograph respectively, record the chromatogram and calculate the recovery rate. The results are shown in Table 11.

[0115] Table 11 Recovery test results

[0116]

[0117]

[0118] The results showed that the average recovery rate of each impurity was between 80% and 120%, and the RSD value was ≤10.0%, indicating that the method had good accuracy.

[0119] Example 6 Durability Test

[0120] Spiked test sample solution: Take an appropriate amount of the product, weigh it accurately, dissolve and dilute it with anhydrous methanol to make a solution containing about 5 mg of the product per 1 ml, as the test sample stock solution, accurately measure 2 ml of the test sample stock solution and 1 ml of the impurity mixed stock solution and place them in the same 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0121] Control solution: Accurately measure 1 ml of the spiked test solution, place it in a 100 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0122] Take blank solution, spiked test solution and control solution, respectively, and analyze under standard chromatographic conditions, column flow rate change ±0.1ml / min, temperature change ±2℃, mobile phase salt concentration change ±2mmol / L, pH value change ±0.1, mobile phase organic phase ratio change ±2%, different batches of chromatographic columns, calculate the RSD of the content of each related substance, and examine the durability. The durability chromatographic conditions are shown in Table 12, and the durability results are shown in Table 13.

[0123] Table 12 Durability Chromatographic Conditions

[0124]

[0125] Table 13 Durability results

[0126]

[0127]

[0128] The results show that under the conditions of flow rate change ±0.1ml / min, temperature change ±2℃, mobile phase pH change ±0.1, organic phase ratio change ±2%, potassium dihydrogen phosphate concentration ±2mmol / L, and different batches of chromatographic columns, the RSD of the content of each related substance is less than 10.0%. It has good durability.

Claims

1. A method for detecting related substances of regorafenib starting material (4-chloro-3-trifluoromethylphenyl isocyanate), characterized in that: The method is a high performance liquid chromatography method, which adopts a reverse chromatographic column, uses a mixed solution of phosphate buffer, acetonitrile and methanol as an eluent, and performs isocratic or gradient elution.

2. The detection method according to claim 1, wherein mobile phase A and / or mobile phase B are used as eluents, wherein mobile phase A is phosphate buffer [10 mmol / L potassium dihydrogen phosphate (phosphoric acid adjusted to pH 2.4)]-acetonitrile (95:5); mobile phase B is acetonitrile-methanol (80:20); the concentration of the phosphate buffer is 8 mmol / L to 12 mmol / L; preferably, the concentration of the phosphate buffer is 10 mmol / L; wherein the phosphate buffer is adjusted to pH 2.3 to 2.5, and the acid is selected from one of formic acid, acetic acid, trifluoroacetic acid, and phosphoric acid, and preferably, the acid is phosphoric acid.

3. The detection method according to claim 1, wherein the reverse phase chromatography column uses a non-polar stationary material as a filler; preferably, the reverse phase chromatography uses phenylsilane bonded silica gel as a filler; more preferably, the reverse phase chromatography column is Kromasil 100-5phenyl, with a specification of 4.6 mm×250 mm, 5 μm.

4. The detection method according to claim 1, wherein the column temperature is 28°C to 32°C; preferably, the column temperature is 30°C.

5. The detection method according to claim 1, wherein the ratio of the mobile phase A is phosphate buffer-acetonitrile is (93-97):(7-3), preferably 95:

5.

6. The detection method according to claim 1, wherein the flow rate of the eluent is 0.7-0.9 ml / min; preferably, the flow rate of the eluent is 0.8 ml / min.

7. The detection method according to claim 1 is performed on a high performance liquid chromatograph using a diode array detector or an ultraviolet detector; preferably, the analysis method is performed on a high performance liquid chromatograph using an ultraviolet detector, and its detection wavelength is 222nm to 242nm; preferably 232nm.

8. A method for detecting 4-chloro-3-(trifluoromethyl)aniline, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]urea, 2-chloro-5-nitro-trifluoromethylbenzene, methyl[(3-trifluoromethyl)phenyl]carbamate, 2-chloro-3-(trifluoromethyl)aniline, and methyl[2-chloro-3-(trifluoromethyl)phenyl]carbamate in 4-chloro-3-trifluoromethylphenyl isocyanate, characterized in that: The method is a high performance liquid chromatography method, which uses a reverse phase chromatography column, a detector is an ultraviolet absorption detector, and a detection wavelength is 246nm; The column temperature was 30°C; Mobile phase A was phosphate buffer [10 mmol / L potassium dihydrogen phosphate (pH adjusted to 2.4 with phosphoric acid)]-acetonitrile (95:5); Mobile phase B was acetonitrile-methanol (80:20) Flow rate: 0.8 ml / min; Perform gradient elution according to the following table:

9. A method for detecting related substances in 4-chloro-3-trifluoromethylphenyl isocyanate, characterized in that: The steps include: (1) Preparation of test solution: Take an appropriate amount of the product, add anhydrous methanol to dissolve and dilute to a solution of appropriate concentration; (2) Preparation of control solution: Accurately measure an appropriate amount of the test solution and dilute it with acetonitrile-water (70:30) to a solution of appropriate concentration; (3) Preparation of system suitability solution: Take appropriate amounts of reference substances such as 4-chloro-3-(trifluoromethyl)aniline, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]urea, 2-chloro-5-nitro-trifluoromethylbenzene, methyl[(3-trifluoromethyl)phenyl]carbamate, 2-chloro-3-(trifluoromethyl)aniline, methyl[2-chloro-3-(trifluoromethyl)phenyl]carbamate, and 4-chloro-3-trifluoromethylphenylisocyanate, and dilute them with acetonitrile-water (70:30) to prepare a solution of appropriate concentration; (4) Determination of test products: Use phenylsilane bonded silica gel as filler [Kromasil 100-5Phenyl (4.6mm×250mm, 5μm) or chromatographic column with equivalent performance]; use 10mmol / L potassium dihydrogen phosphate buffer (pH adjusted to 2.4 with phosphoric acid)-acetonitrile (95:5) as mobile phase A, methanol-acetonitrile (20:80) as mobile phase B, and perform gradient elution according to the following table; flow rate is 0.8ml per minute; column temperature is 30℃; detection wavelength is 246nm; injector temperature is 4℃; injection volume is 5μl. Take an appropriate amount of test solution and / or reference solution, inject it into the liquid chromatograph, and record the chromatographic column chart; (5) Determine the contents of 4-chloro-3-(trifluoromethyl)aniline, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]urea, 2-chloro-5-nitro-trifluoromethylbenzene, methyl[(3-trifluoromethyl)phenyl]carbamate, 2-chloro-3-(trifluoromethyl)aniline, and methyl[2-chloro-3-(trifluoromethyl)phenyl]carbamate in the test sample by the self-control method.

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