High performance liquid chromatography analysis method for 4-chlorobenzoyl chloride and related substances thereof

Abstract

The invention relates to the technical field of medicine quality control, in particular to a high performance liquid chromatography method for 4-chlorobenzoyl chloride and related substances thereof. According to the high performance liquid chromatography analysis method, a reversed-phase high performance liquid chromatography method is adopted, a mobile phase A and a mobile phase B are used as eluents for gradient elution, the mobile phase A is a trifluoroacetic acid solution, and the mobile phase B is methanol. According to the present invention, with the high performance liquid chromatography analysis method, the BZP00575, the BZP00532, the BZP00533, the BZP00534, the BZP00578, the BZP00580, the BZP00579, the BZP00582, the BZP00581, the BZP00583 and the BZP00584 can be simultaneously separated, the 11 related substances can be well separated from the 4-chlorobenzoyl chloride, the sensitivity is high, and the durability is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical quality control, and particularly relates to a high performance liquid chromatography analysis method for 4-chlorobenzoyl chloride and related substances thereof. BACKGROUND

[0002] 4-chlorobenzoyl chloride (English name 4-Chlorobenzoyl chloride) is an organic compound with the chemical formula C7H4Cl2O, which is a colorless or slightly yellow transparent liquid, insoluble in water, soluble in alcohol, ether, and acetone, and easily decomposed in water. It is an important pharmaceutical intermediate and can also be used in the preparation of solvents or pesticides.

[0003] The structural formula of 4-chlorobenzoyl chloride is as follows: .

[0004] The prior art discloses that 11 process impurities, such as compounds BZP00575, BZP00532, BZP00533, BZP00534, BZP00579, BZP00582, BZP00583, BZP00578, BZP00580, BZP00581 and BZP00584, are generated in the production process of 4-chlorobenzoyl chloride, and the structural formulas are shown in Table 1.

[0005] Table 1 Structural formulas of process impurities of 4-chlorobenzoyl chloride

[0006] The detection of related substances is a key indicator for controlling the quality of drugs and drug intermediates. At present, the research on the detection of related substances of 4-chlorobenzoyl chloride is relatively less, which brings great risk to the safety of using 4-chlorobenzoyl chloride as a drug intermediate.

[0007] Chinese patent CN114594193B discloses a liquid phase detection method for detecting 4-chlorobenzoyl chloride. The acyl chloride compound is derivatized with excess anhydrous methanol, and the generated methyl ester compound is detected to achieve the purpose of detecting and controlling m-chlorobenzoyl chloride, o-chlorobenzoyl chloride isomers, and other impurities such as p-chlorobenzoic acid.

[0008] Chinese patent publication CN120142503A discloses a detection method for related substances of 4-chlorobenzoyl chloride. The method uses a chromatographic column with a stationary liquid of 50% phenyl-50% methyl polysiloxane to perform gas chromatography analysis on the 4-chlorobenzoyl chloride sample, achieving accurate detection of four related substances, namely p-chlorobenzoic acid methyl ester, p-chlorobenzoic acid ethyl ester, o-chlorobenzoyl chloride and m-chlorobenzoyl chloride, and their contents in 4-chlorobenzoyl chloride.

[0009] The Chinese patent publication No. CN117471012A discloses a method for detecting 4-chlorobenzoyl chloride related substances by gas chromatography, which can effectively separate the related substances in 4-chlorobenzoyl chloride and quantitatively detect the content of the related substances. Benzoic chloride, m-chlorobenzoyl chloride, 4-chlorobenzoyl chloride, o-chlorobenzoyl chloride, 2,4-dichlorobenzoyl chloride and 3,4-dichlorobenzoyl chloride are sequentially eluted.

[0010] However, the above methods cannot be used to simultaneously detect the 11 process impurity compounds generated in the production process of the above-mentioned 4-chlorobenzoyl chloride raw material.

[0011] Therefore, it is necessary to develop a high-performance liquid chromatography analysis method for 4-chlorobenzoyl chloride and its related substances that can solve the above technical problems. SUMMARY

[0012] The purpose of the present application is to overcome the shortcomings of the prior art and provide a high-performance liquid chromatography analysis method for 4-chlorobenzoyl chloride and its related substances with high separation degree, high sensitivity and / or high durability. The present application has conducted in-depth research on the new analysis and detection method of 4-chlorobenzoyl chloride, and found a high-performance liquid chromatography analysis method that can accurately and effectively separate 11 impurities BZP00575, BZP00532, BZP00533, BZP00534, BZP00579, BZP00582, BZP00583, BZP00578, BZP00580, BZP00581 and BZP00584, thereby achieving the purpose of better controlling the quality of 4-chlorobenzoyl chloride.

[0013] The present application is realized by the following technical solutions: The present application provides a high-performance liquid chromatography analysis method for 4-chlorobenzoyl chloride and its related substances in the first aspect, which adopts a reversed-phase high-performance liquid chromatography method, uses mobile phase A and mobile phase B as eluent for gradient elution, the mobile phase A is a 0.09-0.11% trifluoroacetic acid solution, and the mobile phase B is methanol, and the conditions of the gradient elution include:

[0014] Wherein 43≤m1≤47, 18≤m2≤22, 13≤m3≤17.

[0015] As an embodiment of the present application, the chromatographic column uses octadecylsilane pentafluorophenyl bonded silica gel as the filler.

[0016] Preferably, the chromatographic column is ACE Excel 3 C18-PFP with a size of 4.6mm x 150mm.

[0017] As an embodiment of the present application, the mobile phase A is a 0.09-0.11% trifluoroacetic acid solution, preferably a 0.1% trifluoroacetic acid solution.

[0018] As an embodiment of the present application, the conditions of the gradient elution comprise:

[0019] As an embodiment of the present application, the related substances are impurities BZP00575, BZP00532, BZP00533, BZP00534, BZP00579, BZP00582, BZP00583, BZP00578, BZP00580, BZP00581 and BZP00584, and the chemical structural formulas are shown in Table 1.

[0020] As an embodiment of the present application, the high performance liquid chromatography analysis method comprises the following optional chromatographic conditions: the column temperature of the chromatographic column is 20-40℃; and / or the flow rate of the mobile phase is 0.6-1.0ml / min; and / or the detector is an ultraviolet detector (including but not limited to DAD or VWD detector), and the detection wavelength is 218-242nm; and / or the injection volume is 10-20μl; and / or the temperature of the injector is 4℃-25℃.

[0021] As a preferred embodiment of the present application, the high performance liquid chromatography analysis method comprises the following optional chromatographic conditions: the column temperature of the chromatographic column is 32-37℃, more preferably 32-35℃, and more further preferably 35℃; and / or the flow rate of the mobile phase is 0.7-0.9ml / min, more preferably 0.75-0.85ml / min, and more further preferably 0.8ml / min; and / or the detection wavelength is 240nm and 220nm; and / or the injection volume is 10μl; and / or the temperature of the injector is 4℃.

[0022] As an embodiment of the present application, the high performance liquid chromatography analysis method comprises the following steps: (1) Preparation of the test sample solution: taking 4-chlorobenzoyl chloride sample, dissolving with methanol as the test sample solution; (2) Preparation of the control solution: taking the test sample solution, diluting with methanol as the control solution; (3) Preparation of impurity stock solution: impurity reference substances BZP00532, BZP00533 and BZP00534 were dissolved in methanol to obtain impurity stock solution 1; Impurity reference substances BZP00575, BZP00578, BZP00579, BZP00580, BZP00583 and BZP00582 were dissolved in methanol to obtain impurity stock solution 2; Impurity reference substances BZP00581 and BZP00584 were dissolved in methanol to obtain impurity stock solution 3; Impurity stock solution 1, impurity stock solution 2 and impurity stock solution 3 were mixed in a certain proportion and diluted with methanol to obtain impurity stock solution 4; The preparation of the impurity stock solution can be directly mixing the impurity reference substances and then dissolving in methanol, or dissolving one or more impurity reference substances in methanol and then mixing, as long as it can be completely dissolved to achieve the desired preparation concentration; (4) Preparation of system suitability solution: 4-chlorobenzoyl chloride sample and impurity stock solution 4 were mixed and dissolved in methanol to obtain system suitability solution; (5) Sample detection: the system suitability solution, test sample solution and control solution were injected into a high performance liquid chromatograph to obtain the content of 4-chlorobenzoyl chloride and related substances.

[0023] The content of related substances in the present application was calculated according to the following formula: ; Wherein: A i is the peak area of related substances in the test sample solution; A GD is the peak area of the control solution.

[0024] As a preferred embodiment of the present application, the mass-volume ratio of 4-chlorobenzoyl chloride sample to methanol in step (1) is 35-45 mg:20 ml.

[0025] As a preferred embodiment of the present application, the methanol is diluted to 50-100 times the volume in step (2).

[0026] As a preferred embodiment of the present application, the mass-volume ratio of BZP00532, BZP00533 and BZP00534 to methanol in the impurity stock solution 1 in step (3) is independently selected from 8-12 mg:10 ml; the mass-volume ratio of BZP00575, BZP00578, BZP00579, BZP00580, BZP00583 and BZP00582 to methanol in the impurity stock solution 2 is independently selected from 5-7 mg:10 ml; the mass-volume ratio of BZP00581 and BZP00584 to methanol in the impurity stock solution 3 is independently selected from 5-7 mg:10 ml; the volume ratio of the impurity stock solution 1, the impurity stock solution 2 and the impurity stock solution 3 is 1-3:0.5-2:0.5-2; the volume ratio of the impurity stock solution 1 and the impurity stock solution 4 is 1:4-6.

[0027] As a preferred embodiment of the present application, the mass-volume ratio of the 4-chlorobenzoyl chloride sample and the impurity stock solution 4 in step (4) is 35-45 mg:1 ml; the mass-volume ratio of the 4-chlorobenzoyl chloride sample and the system suitability solution is 1-3 mg:1 ml.

[0028] In the detection process of the high performance liquid chromatography analysis method of the present application, the impurities BZP00575, BZP00532, BZP00533, 4-chlorobenzoyl chloride, BZP00534, BZP00578, BZP00580, BZP00579, BZP00582, BZP00581, BZP00583 and BZP00584 in the system suitability solution (220 nm) are sequentially eluted, and the separation degree (240 nm) of 4-chlorobenzoyl chloride and the adjacent impurities is not less than 1.5.

[0029] The present application has the following beneficial effects: The detection results show that under the high performance liquid chromatography analysis method described in the present application, the impurities BZP00575, BZP00532, BZP00533, BZP00534, BZP00578, BZP00580, BZP00579, BZP00582, BZP00581, BZP00583 and BZP00584 are well separated from 4-chlorobenzoyl chloride, and the peak shape is good, the separation degree (240 nm) of 4-chlorobenzoyl chloride and the adjacent impurities in the system suitability solution is not less than 1.5, the operation is simple, the sensitivity is high, the durability is good, the product quality can be well controlled, and the requirements of the Chinese Pharmacopoeia can be met. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The HPLC chromatogram of the system suitability solution of Example 1 is shown in Figure 1. Figure 2 The partial enlarged view of Figure 1 is shown in Figure 2. Figure 1 The partial enlarged view of Figure 1 is shown in Figure 2. Figure 3 Linear standard curve plot for Example 1 4-chlorobenzoyl chloride; Figure 4 Linear standard curve plot for Example 1 Impurity B ZP00575; Figure 5 Linear standard curve plot for Example 1 Impurity B ZP00532; Figure 6 Linear standard curve plot for Example 1 Impurity B ZP00533; Figure 7 Linear standard curve plot for Example 1 Impurity B ZP00534; Figure 8 Linear standard curve plot for Example 1 Impurity B ZP00578; Figure 9 Linear standard curve plot for Example 1 Impurity B ZP00580; Figure 10 Linear standard curve plot for Example 1 Impurity B ZP00579; Figure 11 Linear standard curve plot for Example 1 Impurity B ZP00582; Figure 12 Linear standard curve plot for Example 1 Impurity B ZP00581; Figure 13 Linear standard curve plot for Example 1 Impurity B ZP00583; Figure 14 Linear standard curve plot for Example 1 Impurity B ZP00584; Figure 15 HPLC chromatogram of the control solution of Example 1; Figure 16 HPLC chromatogram of the system suitability solution of Comparative Example 1; Figure 17 Close-up view of Figure 16 Figure 18 HPLC chromatogram of the system suitability solution of Comparative Example 2; Figure 19 Close-up view of Figure 18 Figure 20 HPLC chromatogram of the system suitability solution of Comparative Example 3; Figure 21 HPLC chromatogram of the system suitability solution of Comparative Example 4; Figure 22 HPLC chromatogram of the system suitability solution of Comparative Example 5; Figure 23 ​​HPLC chromatogram of the positioning solution for each impurity of Comparative Example 6; Figure 24 is a partial enlarged view of Figure 23 DETAILED DESCRIPTION

[0031] The present application will be further described below in connection with specific embodiments, and the advantages and features of the present application will become more apparent with the description. However, these embodiments are only exemplary, and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

[0032] Example 1 1. Chromatographic conditions Instrument: High performance liquid chromatograph: Thermo Fisher U3000, DAD detector; Chromatographic column: ACE Excel 3 C18-PFP (filler is octadecylsilane pentafluorophenyl bonded silica gel), 4.6 mm x 150 mm chromatographic column; Mobile phase A: 0.1% trifluoroacetic acid solution (take 1 ml of trifluoroacetic acid, add 1000 ml of water to dissolve, mix, and ultrasonic, and then obtain); Mobile phase B: methanol; Detection wavelength: 240 nm (BZP00575, BZP00532, BZP00533, 4-chlorobenzoyl chloride, BZP00534, BZP00579, BZP00582, BZP00583, other maximum single impurities), and 220 nm (BZP00578, BZP00580, BZP00581, BZP00584); Flow rate: 0.8 ml / min; Column temperature: 35°C; Injection volume: 10 μl; Injection tray temperature: 4°C.

[0033] The gradient elution program is shown in Table 2.

[0034] Table 2 Gradient elution program of Example 1

[0035] 2. Experimental procedure Take about 40 mg of 4-chlorobenzoyl chloride sample, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, and use as the test solution; ​Accurately take 1 ml of the test solution, place it in a 100-ml flask, dilute to the mark with methanol, shake to mix, and use as the control solution. Accurately weigh about 50 mg of impurity reference substance BZP00532, BZP00533 and BZP00534, place them in a 50-ml flask, dissolve and dilute to the mark with the solvent, shake to mix, and use as impurity stock solution 1.

[0036] Accurately weigh about 60 mg of impurity reference substance BZP00575, BZP00578, BZP00579, BZP00580, BZP00583, BZP00582, place them in a 100-ml flask, dissolve and dilute to the mark with methanol, shake to mix, and use as impurity stock solution 2.

[0037] Accurately weigh about 60 mg of impurity reference substance BZP00581 and BZP00584, place them in a 100-ml flask, dissolve and dilute to the mark with methanol, shake to mix, and use as impurity stock solution 3.

[0038] Accurately take 4 ml of impurity stock solution 1, 2 ml of impurity stock solution 2 and 2 ml of impurity stock solution 3, place them in a 20-ml flask, dilute to the mark with methanol, shake to mix, and use as impurity stock solution 4.

[0039] Accurately weigh about 40 mg of 4-chlorobenzoyl chloride, place it in a 20-ml flask, accurately add 1 ml of impurity stock solution 4, dissolve and dilute to the mark with methanol, shake to mix, and use as the system suitability solution.

[0040] Determination was carried out according to the above conditions. Take 10 μl of the system suitability solution, inject it into the high performance liquid chromatograph. Accurately take 10 μl of the test solution and the control solution, inject them into the high performance liquid chromatograph, and record the chromatogram. The HPLC chromatogram of the system suitability solution is shown in Figure 1 and Figure 2 The detection results show that, under the chromatographic conditions, in the system suitability solution (220 nm), impurities BZP00575, BZP00532, BZP00533, 4-chlorobenzoyl chloride, BZP00534, BZP00578, BZP00580, BZP00579, BZP00582, BZP00581, BZP00583 and BZP00584 eluted in turn, corresponding to peaks No. 1-12, respectively, and the separation degree (240 nm) between 4-chlorobenzoyl chloride and the adjacent impurities is not less than 1.5, which meets the requirements of the Chinese Pharmacopoeia.

[0041] 3. Sensitivity determination Take impurities BZP00575, BZP00532, BZP00533, BZP00534, BZP00578, BZP00580, BZP00579, BZP00582, BZP00581, BZP00583, BZP00584 and 4-chlorobenzoyl chloride control substance, dilute with methanol to prepare a series of different concentrations of solution, respectively, 10 μl of sample, so that the impurities BZP00575, BZP00532, BZP00533, BZP00534, BZP00578, BZP00580, BZP00579, BZP00582, BZP00581, BZP00583 and BZP00584 and 4-chlorobenzoyl chloride as the baseline noise at least 3 times the signal. Through the test, the minimum detection amount of impurities BZP00575, BZP00532, BZP00533, BZP00534, BZP00578, BZP00580, BZP00579, BZP00582, BZP00581, BZP00583 and BZP00584 and 4-chlorobenzoyl chloride is 1.5 ng, 1.5 ng, 3.1 ng, 2.7 ng, 2.9 ng, 2.8 ng, 2.9 ng, 1.5 ng, 2.7 ng, 2.8 ng, 2.6 ng and 1.5 ng (S / N≥3) respectively, if the concentration of 4-chlorobenzoyl chloride is about 2 mg per 1 ml when checking the sample, the detection limit of impurities BZP00575, BZP00532, BZP00533, BZP00534, BZP00578, BZP00580, BZP00579, BZP00582, BZP00581, BZP00583 and BZP00584 and 4-chlorobenzoyl chloride is 0.008%, 0.008%, 0.015%, 0.014%, 0.015%, 0.014%, 0.015%, 0.008%, 0.014%, 0.014%, 0.013% and 0.007% respectively, which can fully meet the determination requirements of 4-chlorobenzoyl chloride and its impurities.

[0042] 4. Solution stability determination Take the control solution, test solution and system suitability solution, place them in the sample tray at 4°C, and detect them at different times. The control solution is stable for at least 65 hours (peak area ratio is between 98% and 102%); the test solution is stable for at least 7 hours (the absolute value of the change in the content of each known impurity, unknown impurity and total impurity is <0.05%); the system suitability solution is stable for at least 70 hours, and the outburst and separation of each impurity can meet the system suitability requirements.

[0043] 5. Linear relationship determination Take about 50 mg of impurity reference substance BZP00532, BZP00533 and BZP00534, accurately weigh, place in a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, as impurity stock solution 1.

[0044] Take about 60 mg of impurity reference substance BZP00575, BZP00578, BZP00579, BZP00580, BZP00583, BZP00582, accurately weigh, place in a 100 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, as impurity stock solution 2.

[0045] Take about 60 mg of impurity reference substance BZP00581 and BZP00584, accurately weigh, place in a 100 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, as impurity stock solution 3.

[0046] Take about 60 mg of 4-chlorobenzoyl chloride, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, as 4-chlorobenzoyl chloride stock solution.

[0047] Accurately take 3 ml of impurity stock solution 1, 5 ml of impurity stock solution 2 and impurity stock solution 3, and 1 ml of 4-chlorobenzoyl chloride stock solution, place in the same 15 ml volumetric flask, dilute to the mark with methanol, shake well, as linear stock solution, accurately take an appropriate amount to prepare a linear solution.

[0048] BZP00575 is linear in the range of 1.0029-30.0884 μg / ml, y = 0.5835x-0.0306, R² = 0.9998, r = 0.9999.

[0049] BZP00532 is linear in the range of 1.0020-30.0609 μg / ml, y = 1.1885x-0.0609, R² = 0.9998, r = 0.9999.

[0050] BZP00533 is linear in the range of 1.0168-30.5051 μg / ml, y = 0.2740x-0.0353, R² = 0.9997, r = 0.9998.

[0051] 4-chlorobenzoyl chloride is linear in the range of 0.9880-29.6406 μg / ml, y = 1.1579x+0.0124, R² = 0.9998, r = 0.9999.

[0052] BZP00534 is linear in the range of 0.9021-27.0644 μg / ml, y = 0.3896x-0.0622, R² = 0.9998, r = 0.9999.

[0053] BZP00578 was linear in the range of 0.9784-29.3524 μg / ml, y = 0.6023x - 0.0292, R² = 0.9997, r = 0.9998.

[0054] BZP00580 was linear in the range of 0.9305-27.9142 μg / ml, y = 0.5997x - 0.0314, R² = 0.9996, r = 0.9998.

[0055] BZP00579 was linear in the range of 0.9814-29.4425 μg / ml, y = 0.5717x - 0.0010, R² = 0.9999, r = 0.9999.

[0056] BZP00582 was linear in the range of 1.0221-30.6628 μg / ml, y = 0.7959x - 0.0588, R² = 0.9999, r = 0.9999.

[0057] BZP00581 was linear in the range of 0.9022-27.0671 μg / ml, y = 0.4821x + 0.0564, R² = 0.9995, r = 0.9998.

[0058] BZP00583 was linear in the range of 0.9472-28.4169 μg / ml, y = 0.3318x + 0.0528, R² = 0.9996, r = 0.9998.

[0059] BZP00584 was linear in the range of 0.8519-25.5584 μg / ml, y = 0.7424x + 0.0514, R² = 0.9998, r = 0.9999. The method was good in linearity. The linear standard curve graph was shown in Figures 3-14 .

[0060] 6. Durability test Under different durability test conditions (see Table 3), 10 μl of system suitability solution was injected into the liquid chromatograph, and the chromatogram was recorded. The separation degree and detection amount between 4-chlorobenzoyl chloride and adjacent impurities in the system suitability solution were investigated, and the results are shown in Tables 4 and 5.

[0061] Table 3 Durability test conditions

[0062] Table 4 Durability test results (1)

[0063] Table 5 Durability test results (2)

[0064] The above experimental results show that the method of the present application is simple and sensitive in operation, good in separation, and good in durability, and thus the method of the present application is good in specificity and reproducibility, and can better control the quality of 4-chlorobenzoyl chloride.

[0065] 7. Sample detection An appropriate amount of 4-chlorobenzoyl chloride samples from three batches were respectively prepared into solutions with a concentration of 2 mg of 4-chlorobenzoyl chloride per 1 ml of methanol, as test sample solutions; sample 1 batch number was 3-20230801, sample 2 batch number was 3-20230703, and sample 3 batch number was 2-20230713; 1 ml of the test sample solution was precisely taken and placed in a 100 ml volumetric flask, diluted to the mark with methanol, and shaken to obtain a control solution. The chromatogram of the control solution of sample 1 is shown in Figure 15 ; the detection results are shown in Table 6 below.

[0066] Table 6 Sample detection results

[0067] Comparative Example 1 The difference from Example 1 is only that the mobile phase B is methanol-acetonitrile (volume ratio of 80:20), and the rest of the conditions are the same as the chromatographic conditions and experimental steps of Example 1, and sample 1 is used. The results are shown in Figure 16 and Figure 17 Impurities BZP00532 and BZP00575 cannot be separated.

[0068] Comparative Example 2 The difference from Example 1 is only that the mobile phase B is methanol-acetonitrile (volume ratio of 90:10), and the rest of the conditions are the same as the chromatographic conditions and experimental steps of Example 1, and sample 1 is used. The results are shown in Figure 18 and Figure 19 Impurities BZP00578 and BZP00581 cannot be separated.

[0069] From the above results, it can be seen that the addition of acetonitrile in the mobile phase B cannot completely separate the impurities, and therefore 100% methanol is used as the mobile phase B to improve the selectivity.

[0070] Comparative Example 3 The difference from Example 1 is only that the gradient elution program is different, as shown in Table 7, and the rest of the conditions are the same as the chromatographic conditions and experimental steps of Example 1, and sample 1 is used. The results are shown in Figure 20As shown, impurity BZP00578 and impurity BZP00581 cannot be separated, and the resolution is 1.04.

[0071] Table 7 Gradient elution procedure of Comparative Example 3

[0072] Comparative Example 4 The difference from Example 1 is only the gradient elution procedure, which is shown in Table 8. The rest of the conditions are the same as the chromatographic conditions and experimental steps of Example 1, and sample 1 is used. The results are shown in Figure 21 As shown, impurity BZP00578 and impurity BZP00581 cannot be separated, and the resolution is 1.15.

[0073] Table 8 Gradient elution procedure of Comparative Example 4

[0074] Comparative Example 5 The difference from Example 1 is only the gradient elution procedure, which is shown in Table 9. The rest of the conditions are the same as the chromatographic conditions and experimental steps of Example 1, and sample 1 is used. The results are shown in Figure 22 As shown, multiple impurities between 7-10 minutes cannot be separated.

[0075] Table 9 Gradient elution procedure of Comparative Example 5

[0076] Comparative Example 6 The difference from Example 1 is only the type of chromatographic column, which is a conventional octadecylsilane-bonded silica gel chromatographic column filler Agilent Edipse XDB-C18, 250 mm x 4.6 mm, 5 μm. The rest of the conditions are the same as the chromatographic conditions and experimental steps of Example 1, and the positioning solution of each impurity is used. The results are shown in Figure 23 and Figure 24 The results show that the retention and separation of multiple impurities are poor, indicating that the conventional C18 chromatographic column has poor selectivity and is difficult to separate the 11 related substance impurities of the present application.

[0077] The positioning solution of each impurity BZP00532, BZP00533, BZP00575, BZP00534, BZP00482, BZP00580, BZP00579, BZP00581, BZP00582, BZP00583 and BZP00584 is prepared: an appropriate amount of each of the above impurity reference substances is separately weighed into different 10 ml volumetric flasks, dissolved and diluted with methanol to prepare a positioning solution containing about 0.1 mg of impurity reference substance per 1 ml, which is used for the positioning of each impurity reference substance.

[0078] Figure 23 and Figure 24 The chromatogram overlay of each impurity reference standard shown from left to right in the figure is the order of locating peaks of impurities BZP00532, BZP00533, BZP00575, BZP00534, BZP00482, BZP00580, BZP00579, BZP00581, BZP00582, BZP00583 and BZP00584, respectively. It can be seen that the multiple impurities eluted in the front (before 11 minutes) are not separated.

[0079] Accuracy test of test example 1 The accuracy is obtained by measuring the recovery rate of the three different concentrations of each impurity to be tested, i.e. the limit of quantification (LOQ), 100% and 150% of the limit. The known amount of impurity is added, and the ratio (recovery rate) between the measured value and the theoretical value of each impurity in the spiked sample is expressed in percentage (%).

[0080] Solution preparation: Impurity stock solution 1: The preparation method is the same as that of Example 1.

[0081] Impurity stock solution 2: The preparation method is the same as that of Example 1.

[0082] Impurity stock solution 3: The preparation method is the same as that of Example 1.

[0083] Impurity stock solution 4: The preparation method is the same as that of Example 1.

[0084] Reference solution: 1 ml of impurity stock solution 4 was accurately measured and placed in a 20 ml volumetric flask, dissolved and diluted to the mark with methanol, and shaken well.

[0085] Recovery rate - low concentration stock solution: 2 ml of impurity stock solution 1, 1 ml of impurity stock solution 2 and 1 ml of impurity stock solution 3 were accurately measured and placed in a 50 ml volumetric flask, diluted to the mark with methanol, and shaken well.

[0086] Background solution: About 40 mg of 4-chlorobenzoyl chloride sample was accurately weighed and placed in a 20 ml volumetric flask, dissolved and diluted to the mark with methanol, and shaken well.

[0087] Low concentration solution (70%): About 40 mg of 4-chlorobenzoyl chloride sample was accurately weighed and placed in a 20 ml volumetric flask, 0.7 ml of impurity stock solution 4 was accurately added, dissolved and diluted to the mark with methanol. Three were prepared by the same method, and were recorded as low-1, low-2 and low-3, respectively. This low concentration solution was used to detect the recovery rates of impurities BZP00532, BZP00534, BZP00578, BZP00580, BZP00579, BZP00582 and BZP00581.

[0088] Low concentration solution (20%): Take 4-chlorobenzoyl chloride sample about 40 mg, accurately weighed, placed in a 20 ml volumetric flask, accurately add the recovery rate-low concentration stock solution 1 ml, add methanol to dissolve and dilute to the mark, shake well. Three are prepared in the same way, respectively, low-4, low-5, low-6. This low concentration solution is used to detect the recovery rate of impurities BZP00575, BZP00533, BZP00583 and BZP00584.

[0089] Medium concentration solution: Take 4-chlorobenzoyl chloride sample about 40 mg, accurately weighed, placed in a 20 ml volumetric flask, accurately add the impurity stock solution 4 1 ml, add methanol to dissolve and dilute to the mark. Three are prepared in the same way, respectively, medium-1, medium-2, medium-3.

[0090] High concentration solution: Take 4-chlorobenzoyl chloride sample about 40 mg, accurately weighed, placed in a 20 ml volumetric flask, accurately add the impurity stock solution 4 1.5 ml, add methanol to dissolve and dilute to the mark. Three are prepared in the same way, respectively, high-1, high-2, high-3.

[0091] The detection was carried out according to the method of Example 1, and the results are shown in Tables 10-20.

[0092] Table 10 Recovery rate results of impurity BZP00575

[0093] Table 11 Recovery rate results of impurity BZP00532

[0094] Table 12 Recovery rate results of impurity BZP00533

[0095] Table 13 Recovery rate results of impurity BZP00534

[0096] Table 14 Recovery rate results of impurity BZP00578

[0097] Table 15 Recovery rate results of impurity BZP00580

[0098] Table 16 Recovery rate results of impurity BZP00579

[0099] Table 17 Recovery rate results of impurity BZP00582

[0100] Table 18 Recovery results of impurity BZP00581

[0101] Table 19 Recovery results of impurity BZP00583

[0102] Table 20 Recovery results of impurity BZP00584

[0103] Conclusion: The recovery rate of BZP00575 was 97.5%~107.5%, the average recovery rate of low concentration was 99.5%, the RSD was 2.1%, the average recovery rate of medium concentration was 107.0%, the RSD was 0.6%, the average recovery rate of high concentration was 104.2%, the RSD was 3.1%, the average recovery rate of nine samples was 103.6%, and the RSD was 3.7%.

[0104] The recovery rate of BZP00532 was 102.5%~116.1%, the average recovery rate of low concentration was 115.1%, the RSD was 1.1%, the average recovery rate of medium concentration was 105.8%, the RSD was 1.2%, the average recovery rate of high concentration was 103.4%, the RSD was 1.2%, the average recovery rate of nine samples was 108.1%, and the RSD was 5.1%.

[0105] The recovery rate of BZP00533 was 99.6%~121.5%, the average recovery rate of low concentration was 116.3%, the RSD was 5.8%, the average recovery rate of medium concentration was 101.0%, the RSD was 1.6%, the average recovery rate of high concentration was 102.6%, the RSD was 2.0%, the average recovery rate of nine samples was 106.6%, and the RSD was 7.7%.

[0106] The recovery rate of BZP00534 was 87%~92.8%, the average recovery rate of low concentration was 90.6%, the RSD was 2.3%, the average recovery rate of medium concentration was 90.3%, the RSD was 3.3%, the average recovery rate of high concentration was 90.6%, the RSD was 1.5%, the average recovery rate of nine samples was 90.5%, and the RSD was 2.2%.

[0107] The recovery rate of BZP00578 was 100.9%~110.2%, the average recovery rate of low concentration was 105.1%, the RSD was 3.6%, the average recovery rate of medium concentration was 107.6%, the RSD was 4.1%, the average recovery rate of high concentration was 104.9%, the RSD was 2.5%, the average recovery rate of nine samples was 105.9%, and the RSD was 3.3%.

[0108] The above detailed description is a specific description of one of the possible embodiments of the present application, which is not used to limit the patent scope of the present application, and any equivalent implementation or change made without departing from the present application shall be included in the scope of the technical solutions of the present application.

Claims

1. A method for the analysis of 4-chlorobenzoyl chloride and its related substances by high performance liquid chromatography, characterized in that, The gradient elution is performed by using mobile phase A and mobile phase B as eluent, the mobile phase A is trifluoroacetic acid solution, the mobile phase B is methanol, and the gradient elution conditions include: ； Wherein, 43≤m1≤47, 18≤m2≤22, 13≤m3≤17.

2. The high performance liquid chromatography analysis method according to claim 1, characterized in that, The chromatographic column uses octadecylsilane pentafluorophenyl bonded silica gel as the filler.

3. The high performance liquid chromatography method of claim 2, wherein, The chromatographic column is ACE Excel 3 C18-PFP, with a size of 4.6mm*150mm.

4. The high performance liquid chromatography analysis method of claim 1, wherein, The mobile phase A is 0.09-0.11% trifluoroacetic acid solution.

5. The method of high performance liquid chromatography according to claim 1, wherein, The gradient elution conditions include: 。 6. The high performance liquid chromatography analysis method of claim 1, wherein, The related substances are impurities BZP00575, BZP00532, BZP00533, BZP00534, BZP00579, BZP00582, BZP00583, BZP00578, BZP00580, BZP00581 and BZP00584, and the chemical structural formula is as follows: 。 7. The method of high performance liquid chromatography according to claim 1, wherein, The high performance liquid chromatography analysis method comprises the following optional chromatographic conditions: The column temperature of the chromatographic column is 20-40℃; and / or The flow rate of the mobile phase is 0.6-1.0ml / min; and / or The detector uses an ultraviolet detector, and the detection wavelength is 218-242nm; and / or The injection volume is 10-20μl; and / or The injector temperature is 4℃-25℃.

8. The method of high performance liquid chromatography according to claim 7, wherein, The high performance liquid chromatography analysis method comprises the following optional chromatographic conditions: The column temperature of the chromatographic column is 32-37℃; and / or The flow rate of the mobile phase is 0.7-0.9ml / min; and / or The detection wavelength is 240nm and 220nm; and / or The injection volume is 10μl; and / or The injector temperature is 4℃.

9. The high performance liquid chromatography method according to any one of claims 1 to 8, wherein, The high performance liquid chromatography analysis method comprises the following steps: (1) Preparation of test solution: take 4-chlorobenzoyl chloride sample, dissolve in methanol as test solution; (2) Preparation of control solution: take the test solution, dilute with methanol as control solution; (3) Preparation of impurity stock solution: take impurity reference substance BZP00532, BZP00533 and BZP00534, dissolve in methanol as impurity stock solution 1; Take impurity reference substance BZP00575, BZP00578, BZP00579, BZP00580, BZP00583, BZP00582, dissolve in methanol as impurity stock solution 2; Take impurity reference substance BZP00581 and BZP00584, dissolve in methanol as impurity stock solution 3; Mix impurity stock solution 1, impurity stock solution 2 and impurity stock solution 3 according to a certain proportion, dilute with methanol as impurity stock solution 4; (4) Preparation of system suitability solution: mix 4-chlorobenzoyl chloride sample and impurity stock solution 4, dissolve in methanol as system suitability solution; (5) Sample detection: inject system suitability solution, test solution and control solution into high performance liquid chromatograph, and the content of 4-chlorobenzoyl chloride and its related substances is obtained.

10. The method of high performance liquid chromatography according to claim 9, wherein, The mass-volume ratio of 4-chlorobenzoyl chloride sample and methanol in step (1) is 35-45mg:20ml; Diluted to 50-100 times volume with methanol in step (2); The mass-volume ratio of BZP00532, BZP00533, BZP00534 to methanol in the impurity stock solution 1 is independently selected from 8-12 mg:10 ml; the mass-volume ratio of BZP00575, BZP00578, BZP00579, BZP00580, BZP00583, BZP00582 to methanol in the impurity stock solution 2 is independently selected from 5-7 mg:10 ml; the mass-volume ratio of BZP00581 and BZP00584 to methanol in the impurity stock solution 3 is independently selected from 5-7 mg:10 ml; the volume ratio of the impurity stock solution 1, the impurity stock solution 2 and the impurity stock solution 3 is 1-3:0.5-2:0.5-2; the volume ratio of the impurity stock solution 1 and the impurity stock solution 4 is 1:4-6; The mass-volume ratio of the 4-chlorobenzoyl chloride sample and the impurity stock solution 4 in step (4) is 35-45 mg:1 ml; the mass-volume ratio of the 4-chlorobenzoyl chloride sample and the system suitability solution is 1-3 mg:1 ml.

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