HPLC quantitative detection method for o-methylbenzyl chloride and impurities thereof

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to an HPLC (High Performance Liquid Chromatography) quantitative detection method for o-methylbenzyl chloride and impurities thereof. The method comprises the following steps: preparing a blank solution, a test solution and a reference solution, respectively injecting the blank solution, the test solution and the reference solution into a liquid chromatograph, recording chromatograms, and calculating according to a peak area normalization method to obtain the content of o-methylbenzyl chloride in the test solution. The content of o-xylylene dichloride and / or o-xylene in the test solution is calculated according to the peak area by an external standard method, and the content of other impurities in the test solution is calculated according to a main component self-contrast method. The separation degree of the main peak and each impurity peak is good, and the precision, the accuracy, the stability, the durability and the reproducibility are all excellent. And a detection technology is provided for quality control of o-methylbenzyl chloride.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to an HPLC quantitative detection method for o-methylbenzyl chloride and its impurities. Background Technology

[0002] o-Methylbenzyl chloride, chemically known as 2-methylbenzyl chloride, has the molecular formula C8H9Cl and a molecular weight of 140.61. Its structural formula is as follows: o-methylbenzyl chloride is a colorless or pale yellow liquid and is the starting material for LXH-1211, an innovative drug used to treat pulmonary arterial hypertension. It is also an important raw material or intermediate for the synthesis of drugs such as the nonsteroidal anti-inflammatory drug ibuprofen, the antihistamine chlorpheniramine, and the antifungal drug clotrimazole derivatives.

[0003] The o-methylbenzyl chloride preparation process uses o-xylene as the starting material, which reacts with chlorine to obtain the product. The reaction may contain process impurities such as o-xylene residue or o-dichlorobenzyl byproduct.

[0004] The level of process impurities present during the synthesis and preparation of o-methylbenzyl chloride plays a crucial role in the quality of the final drug products prepared from o-methylbenzyl chloride as a starting material or intermediate. Therefore, quality control of o-methylbenzyl chloride is particularly important. Patent CN119500024A discloses a production process and system for o-methylbenzyl chloride. To date, there is limited literature on detection methods for o-methylbenzyl chloride. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide an HPLC quantitative detection method for o-methylbenzyl chloride and its impurities. This method exhibits good separation between the main peak and each impurity peak, as well as excellent precision, accuracy, stability, robustness, and reproducibility. It provides an analytical means for the quality control of o-methylbenzyl chloride, thereby more effectively controlling the quality of pharmaceutical end products obtained from o-methylbenzyl chloride as a raw material or intermediate.

[0006] The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities described in this invention involves preparing a blank solution, a test solution, and a reference solution. The blank solution, test solution, and reference solution are injected into a liquid chromatograph, and chromatograms are recorded. The content of o-methylbenzyl chloride in the test solution is calculated using the peak area normalization method. The content of impurities o-dichlorobenzyl and / or o-xylene in the test solution is calculated using the external standard method based on peak area. The content of other impurities in the test solution is calculated using the main component self-comparison method. The structural formula of o-methylbenzyl chloride is as follows: ; The structural formula of o-dichlorobenzyl is as follows: ; The structural formula of o-xylene is as follows: ; The chromatographic conditions in the HPLC method include the mobile phase; the mobile phase is an aqueous solution of phosphoric acid as mobile phase A and methanol as mobile phase B. The chromatographic conditions also include a gradient elution program, which is as follows: 0-2 minutes, mobile phase A accounts for 35-45% of the total mobile phase volume, and mobile phase B accounts for 55-65% of the total mobile phase volume; 2-15 minutes, mobile phase A accounts for 25-35% of the total mobile phase volume, and mobile phase B accounts for 65-75% of the total mobile phase volume; 15-20 minutes, mobile phase A accounts for 25-35% of the total mobile phase volume, and mobile phase B accounts for 65-75% of the total mobile phase volume; 20-25 minutes, mobile phase A accounts for 35-45% of the total mobile phase volume, and mobile phase B accounts for 55-65% of the total mobile phase volume; 25-30 minutes, mobile phase A accounts for 35-45% of the total mobile phase volume, and mobile phase B accounts for 55-65% of the total mobile phase volume.

[0007] The test solution is prepared by weighing an o-methylbenzyl chloride sample, dissolving and diluting it with a solvent to prepare the test solution; the concentration of o-methylbenzyl chloride in the test solution is 0.4-0.6 mg / mL.

[0008] The reference solution is prepared by diluting the stock solution of any single component or mixture of o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride with a solvent to prepare the reference solution; the concentration of any single component in the solution of any single component or mixture of o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride is 0.25-7.5 μg / mL.

[0009] The preparation method of the stock solution is as follows: weigh o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride reference standards respectively, dissolve and dilute them with solvent to prepare a stock solution containing any single component or mixture of o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride; the concentration of any single component in the solution of any single component or mixture of o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride in the stock solution is 2.5-1200 μg / mL.

[0010] The solvent is acetonitrile (anhydrous sodium sulfate or water removed by molecular sieve).

[0011] The mobile phase A is a 0.05 wt.% to 0.2 wt.% aqueous solution of phosphoric acid.

[0012] The parameters and conditions for the liquid chromatography include: the chromatographic column is a C18 column with octadecylsilane-bonded silica gel as the packing material, and the column temperature is 38-42℃.

[0013] The parameters for the liquid chromatography include: the detector is a DAD / PDA detector or an ultraviolet detector, and the detection wavelength is 218-222 nm.

[0014] The total flow rate of mobile phase A and mobile phase B is 0.95-1.05 mL / min.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The HPLC method of the present invention uses octadecylsilane bonded silica gel as the stationary phase. The polarity of the stationary phase is less than that of the mobile phase. That is, it is a reversed-phase high-performance liquid chromatography method, which can simultaneously detect the content of the main component o-methylbenzyl chloride and its impurities.

[0016] (2) The HPLC method of the present invention meets the requirements for the separation of o-methylbenzyl chloride and its impurities o-dichlorobenzyl, o-xylene, etc., and has high accuracy and precision, good stability and reproducibility.

[0017] (3) The HPLC method of the present invention is simple and easy to implement, and facilitates the quality control of raw materials or intermediates such as o-methylbenzyl chloride for pharmaceutical products such as LXH-1211 and ibuprofen during the production process. Attached Figure Description

[0018] Figure 1 This is the HPLC chromatogram of the reference solution from Example 1.

[0019] Figure 2 This is the HPLC chromatogram of the blank solution from Example 1.

[0020] Figure 3 This is the HPLC chromatogram of the mixed solution from Example 1.

[0021] Figure 4 This is the HPLC chromatogram of the o-dichlorobenzyl positioning solution from Example 1.

[0022] Figure 5 The image shows the HPLC chromatogram of the o-xylene localization solution from Example 1.

[0023] Figure 6 This is the HPLC chromatogram of the test solution in Example 1.

[0024] Figure 7 The HPLC chromatogram of the test solution in Example 11 is shown. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments.

[0026] Unless otherwise specified, all raw materials used in the examples were commercially available.

[0027] All instruments and reagents used in the following examples are commercially available products. Methanol: Merck, HPLC grade; Acetonitrile: Merck, HPLC grade; Phosphoric acid: GENERAL-REAGENT, AR grade; Anhydrous sodium sulfate: Wako Pure Chemical Industries, Ltd., AR grade; 5A molecular sieve: Comio, AR grade.

[0028] o-methylbenzyl chloride: Shandong Xinhua Pharmaceutical Co., Ltd., preparation method: o-xylene raw material is pumped into an evaporation kettle and heated to 60±10℃. Chlorine gas (chlorine flow rate 0.35±0.15mol / h) is introduced under visible light catalysis to carry out a gas-phase substitution reaction, producing crude o-methylbenzyl chloride, a small amount of o-dichlorobenzyl chloride, and hydrogen chloride. The generated o-methylbenzyl chloride, o-dichlorobenzyl chloride, and a small amount of unreacted o-xylene are in liquid state and flow back into the evaporation kettle. The crude benzyl chloride in the kettle is then transferred to a distillation kettle, heated to 140℃, and the small amount of unreacted o-xylene in the crude benzyl chloride is distilled off as raw material for the next batch. The temperature is raised to 180±10℃ to distill off o-methylbenzyl chloride, and then distilled off to obtain o-methylbenzyl chloride and a small amount of o-dichlorobenzyl chloride, respectively. The hydrogen chloride gas generated in the reaction enters the hydrogen chloride absorption section. The reaction formula is shown in Equation I below:

[0029] Formula I.

[0030] Example 1 Specificity Test (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0031] Reference stock solution: Accurately weigh the corresponding amount of reference standard according to Table 1, place it in the corresponding volumetric bottle, add solvent to dissolve and dilute to the mark, and shake well.

[0032] Table 1 Weighing and concentration data of the reference standard stock solution

[0033] Impurity mixed stock solution: Accurately measure 1.0 ml of o-dichlorobenzyl and o-xylene reference stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0034] Positioning solution: Measure the corresponding amount of reference standard stock solution according to Table 2, place it in the corresponding volumetric flask, dilute with solvent to the mark, and shake well.

[0035] Table 2 Weighing and concentration data of the positioning solution

[0036] Reference solution: Accurately measure 0.5 ml each of o-methylbenzyl chloride, o-dichlorobenzyl chloride and o-xylene reference stock solutions into a 100 ml volumetric flask, dilute to the mark with solvent and mix well.

[0037] Test solution: Weigh 10.259 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0038] Mixed solution: Accurately weigh 10.365 mg of o-methylbenzyl chloride test sample, place it in a 20 ml volumetric flask, add solvent to dissolve it, accurately measure 2.0 ml of the above impurity mixed stock solution and add it, dilute to the mark with solvent, and shake well.

[0039] (2) Chromatographic conditions: Instrument: Shimadzu LC-20AT high performance liquid chromatograph with PDA detector; Column: Agilent ZORBAX SB-C18 4.6×250mm 5μm; Detection wavelength: 220nm; Mobile phase A: 0.1% aqueous phosphoric acid solution; Mobile phase B: Methanol; Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL; The gradient elution procedure is shown in Table 3; Table 3 Gradient elution program

[0040] (3) Determination: Accurately measure blank solution, reference solution, positioning solution, test solution, and mixed solution and inject them into the liquid chromatograph respectively, and record the chromatograms, such as... Figure 1-6 As shown, Figure 1 This is the HPLC chromatogram of the reference solution from Example 1. Figure 2 This is the HPLC chromatogram of the blank solution from Example 1. Figure 3 This is the HPLC chromatogram of the mixed solution from Example 1. Figure 4 This is the HPLC chromatogram of the o-dichlorobenzyl positioning solution from Example 1. Figure 5 This is the HPLC chromatogram of the o-xylene localization solution from Example 1. Figure 6 This is the HPLC chromatogram of the test solution in Example 1.

[0041] The blank solution showed no significant interference at the main peak of o-methylbenzyl chloride and the peaks of o-dichlorobenzyl and o-xylene. The retention times of each impurity with the main peak of o-methylbenzyl chloride and their relative retention times with the main peak are shown in Table 4 below.

[0042] Table 4. Retention times of each impurity and the main peak of o-methylbenzyl chloride, and their relative retention times.

[0043] The retention times of the main peaks in the o-methylbenzyl chloride test solution and the reference solution were consistent; there was no interference near the main peak and the known impurity peaks in both the test solution and the mixed solution; in the mixed solution chromatogram, the minimum resolution between the known impurities and adjacent peaks, and between the main component and adjacent peaks, was 2.1, and the peak purity of each impurity and o-methylbenzyl chloride met the requirements.

[0044] Example 2 Repeatability Test (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0045] Reference stock solution: Accurately weigh the corresponding amount of reference standard according to Table 5, place it in the corresponding volumetric bottle, add solvent to dissolve and dilute to the mark, and shake well.

[0046] Table 5 Weighing and concentration data of the reference standard stock solution

[0047] Impurity mixed stock solution: Accurately measure 1.0 ml of o-dichlorobenzyl and o-xylene reference stock solution, place it in a 20 ml volumetric flask, add solvent to dilute to the mark, and mix well.

[0048] System suitability solution: Weigh 10.365 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve it, accurately measure 2.0 ml of impurity mixed stock solution, dilute to the mark with solvent, and shake well.

[0049] Reference solutions: Accurately measure 0.5 ml each of o-methylbenzyl chloride, o-dichlorobenzyl chloride, and o-xylene reference stock solutions, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0050] Repeatability test solution: Accurately weigh 10.190 mg, 10.177 mg, 10.190 mg, 10.185 mg, 10.184 mg, and 10.191 mg of the o-methylbenzyl chloride test sample, and place them in separate 20 ml volumetric flasks. Dissolve the solutions in solvent, add 2.0 ml of the impurity stock solution, dilute to the mark with solvent, and shake well to prepare the repeatability test solution. Prepare 6 solutions using the same method.

[0051] (2) Determination: Using an Agilent 1260 high performance liquid chromatograph equipped with a DAD detector, the above system suitability solution, reference solution, blank solution and repeatability test solution were injected according to the chromatographic conditions of Example 1, and the chromatograms were recorded.

[0052] The repeatability test results for o-dichlorobenzyl are shown in Table 6, the repeatability test results for o-xylene are shown in Table 7, and the repeatability test results for unknown impurities - RRT1.07, RRT1.29, RRT1.68, RRT1.72 and total impurities are shown in Table 8.

[0053] Table 6. Repeatability test results of o-dichlorobenzyl

[0054] Table 7 Results of repeatability tests for o-xylene

[0055] Table 8. Repeatability test results for unknown impurities - RRT1.07, RRT1.29, RRT1.68, RRT1.72 and total impurities

[0056] (3) Results: The RSD of the peak area of ​​o-dichlorobenzyl in the reference solution was 0.71%, the RSD of the peak area of ​​o-xylene was 1.36%, and the RSD of the peak area of ​​o-methylbenzyl chloride was 0.58%. Among the 6 repeatability test solutions, the range of o-dichlorobenzyl content was 0.01%, the range of o-xylene content was 0.01%, the maximum range of other single impurities was 0.01%, and the range of total impurities was 0.01%. All of the above met the requirements.

[0057] Example 3 Intermediate Precision Test The lab technician was changed, and the precision solution was re-prepared and tested on different dates.

[0058] (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0059] Reference stock solution: Accurately weigh the corresponding amount of reference standard according to Table 9, place it in the corresponding volumetric bottle, add solvent to dissolve and dilute to the mark, and shake well.

[0060] Table 9 Weighing and concentration data of the reference standard stock solution

[0061] Impurity mixed stock solution: Accurately measure 1.0 ml of o-dichlorobenzyl and o-xylene reference stock solution, place it in a 20 ml volumetric flask, add solvent to dilute to the mark, and mix well.

[0062] System suitability solution: Weigh 10.432 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve it, accurately measure 2.0 ml of impurity mixed stock solution, dilute to the mark with solvent, and shake well.

[0063] Reference solutions: Accurately measure 0.5 ml each of o-methylbenzyl chloride, o-dichlorobenzyl chloride, and o-xylene reference stock solutions, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0064] Intermediate precision test solution: Accurately weigh 10.432 mg, 10.488 mg, 10.508 mg, 10.441 mg, 10.489 mg, and 10.483 mg of the o-methylbenzyl chloride test sample, and place them separately in 20 ml volumetric flasks. Dissolve them in solvent, add 2.0 ml of impurity stock solution, dilute to the mark with solvent, and shake well to prepare the intermediate precision test solution. Prepare 6 solutions using the same method.

[0065] (2) Determination: A Shimadzu LC-20AT high performance liquid chromatograph with a PDA detector was used. According to the chromatographic conditions in Example 1, the above system suitability solution, reference solution, blank solution and intermediate precision test solution were injected and the chromatograms were recorded.

[0066] The intermediate precision test results for o-dichlorobenzyl are shown in Table 10, the intermediate precision test results for o-xylene are shown in Table 11, and the intermediate precision test results for unknown impurities - RRT1.07, RRT1.29, RRT1.68, RRT1.72 and total impurities are shown in Table 12.

[0067] Table 10. Intermediate Precision Test Results of o-Dichlorobenzyl

[0068] Table 11 Results of intermediate precision test for o-xylene

[0069] Table 12 Intermediate precision test results for unknown impurities - RRT1.07, RRT1.28, RRT1.67, RRT1.71 and total impurities

[0070] (3) Results: In the reference solution: the RSD of the peak area of ​​o-dichlorobenzyl was 0.30%, the RSD of the peak area of ​​o-xylene was 0.31%, and the RSD of the peak area of ​​o-methylbenzyl chloride was 0.28%; in the 6 intermediate precision test solutions, the range of o-dichlorobenzyl content was 0, the range of o-xylene content was 0.01%, the maximum range of other single impurities was 0, and the range of total impurities was 0.02%; in the repeatability test of 12 determinations of each target impurity content, the range of o-dichlorobenzyl content was 0.04%, the range of o-xylene content was 0.02%, the maximum range of other single impurities was 0.03%, and the range of total impurities was 0.11%. All of the above meet the requirements.

[0071] Example 4: Limit of Detection and Limit of Quantification Test (1) Solution preparation Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0072] Reference stock solutions: Accurately weigh approximately 5 mg each of o-dichlorobenzyl, o-xylene, and o-methylbenzyl chloride reference standards, place them separately in 5 ml volumetric flasks, dissolve and dilute to the mark with solvent, and mix well to prepare the stock solutions for each reference standard. The specific sample amounts are shown in Table 13 below. Prepare 6 solutions using the same method.

[0073] Stock solutions for the limit of quantitation: Accurately measure 0.5 ml each of o-dichlorobenzyl, o-xylene, and o-methylbenzyl chloride reference stock solutions, place them in a 100 ml volumetric flask, dilute to the mark with solvent, mix well, and use as the stock solutions for the limit of quantitation.

[0074] Limit of Quantitation (LOQ) Solution: Accurately measure 0.5 ml of the LOQ stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and mix well. Prepare 6 aliquots using the same method.

[0075] Detection limit solution: Accurately measure 5.0 ml of the quantitation limit solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and mix well. Prepare 6 aliquots using the same method.

[0076] (2) Determination: Using an Agilent 1260 high performance liquid chromatograph equipped with a DAD detector, blank solution, limit of quantitation solution and limit of detection solution were injected according to the chromatographic conditions of Example 1, and the chromatograms were recorded.

[0077] Calculation formula: Limit of detection (%) = [Limit of detection concentration (µg / ml) / Sample concentration (µg / ml)] × 100%; Limit of quantitation (%) = [Limit of quantitation concentration (µg / ml) / sample concentration (µg / ml)] × 100%.

[0078] The results of the limit of detection and limit of quantitation tests are shown in Table 13.

[0079] Table 13 Results of Limit of Detection and Limit of Quantitation Tests

[0080] (3) Results: The detection limit of o-dichlorobenzyl was 0.1244 μg / ml, the detection limit was 0.025%, and the minimum signal-to-noise ratio was 12; the quantitation limit was 0.2488 μg / ml, the quantitation limit was 0.05%, and the minimum signal-to-noise ratio was 23; the peak area RSD of the quantitation limit was 2.53%; the detection limit of o-xylene was 0.1243 μg / ml, the detection limit was 0.025%, and the minimum signal-to-noise ratio was 5; the quantitation limit was... The concentration of o-methylbenzyl chloride was 0.2485 μg / ml, with a limit of quantitation (LOQ) of 0.05% and a minimum signal-to-noise ratio (SNR) of 11; the peak area RSD of the LOQ was 5.11%. The concentration of o-methylbenzyl chloride was 0.1269 μg / ml, with a LOQ of 0.025% and a minimum SNR of 16; the concentration of o-methylbenzyl chloride was 0.2538 μg / ml, with a LOQ of 0.05% and a minimum SNR of 25; the peak area RSD of the LOQ was 5.32%. All met the requirements.

[0081] Example 5 Linearity Test 1. Content linearity (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0082] Content linearity test solution ①: Weigh 8.214 mg, 8.200 mg and 8.193 mg of o-methylbenzyl chloride reference standard accurately, place them in 20 ml volumetric flasks respectively, add solvent to dissolve and dilute to the mark, shake well, and prepare 3 portions in the same way.

[0083] Content linearity test solution ②: Weigh 9.006 mg, 9.026 mg and 9.022 mg of o-methylbenzyl chloride reference standard accurately, place them in 20 ml volumetric flasks respectively, add solvent to dissolve and dilute to the mark and shake well. Prepare 3 portions in the same way.

[0084] Content linearity test solution ③: Weigh 10.386 mg, 10.409 mg and 10.406 mg of o-methylbenzyl chloride reference standard accurately, place them in 20 ml volumetric flasks respectively, add solvent to dissolve and dilute to the mark, shake well, and prepare 3 portions in the same way.

[0085] Content linearity test solution ④: Weigh 11.162 mg, 11.166 mg and 11.117 mg of o-methylbenzyl chloride reference standard accurately, place them in 20 ml volumetric flasks respectively, add solvent to dissolve and dilute to the mark and shake well. Prepare 3 portions in the same way.

[0086] Content linearity test solution ⑤: Weigh 12.476 mg, 12.435 mg and 12.405 mg of o-methylbenzyl chloride reference standard accurately, place them in 20 ml volumetric flasks respectively, add solvent to dissolve and dilute to the mark, shake well, and prepare 3 portions in the same way.

[0087] (2) Determination: Using an Agilent 1260 high performance liquid chromatograph equipped with a DAD detector, the blank solution and linear test solution were injected according to the chromatographic conditions of Example 1, and the chromatograms were recorded. The linear regression equation was calculated with the average peak area as the abscissa and the average concentration as the ordinate.

[0088] 2. Linearity of related substances (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0089] Linear stock solutions: Accurately weigh approximately 5 mg each of o-dichlorobenzyl, o-xylene, and o-methylbenzyl chloride reference standards, place them separately in 5 ml volumetric flasks, dissolve and dilute to the mark with solvent, and mix well. These are the stock solutions for each reference standard. Specific sample amounts are shown in Table 14 below. Accurately measure 1.0 ml each of the o-dichlorobenzyl, o-xylene, and o-methylbenzyl chloride reference standard stock solutions, place them in 20 ml volumetric flasks, dilute to the mark with solvent, and mix well. This is the linear stock solution. Prepare three aliquots of the linear stock solution using the same method.

[0090] Table 14 Sample Weights of Each Reference Standard for Linear Stock Solution

[0091] Linearity test solution ①: Accurately measure 0.5 ml of the stock solutions of o-dichlorobenzyl, o-xylene, and o-methylbenzyl chloride reference standards, place them in a 100 ml volumetric flask, add solvent to dilute to the mark, mix well, and use as the stock solution for the limit of quantitation (LOQ). Accurately measure 0.5 ml of the LOQ stock solution, place it in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 3 aliquots using the same method.

[0092] Linearity test solution ②: Accurately measure 0.3 ml of linearity stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and prepare 3 portions in the same way.

[0093] Linearity test solution ③: Accurately measure 0.5 ml of linearity stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and prepare 3 portions in the same way.

[0094] Linearity test solution ④: Accurately measure 1.0 ml of linearity stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and prepare 3 portions in the same way.

[0095] Linearity test solution ⑤: Accurately measure 1.5 ml of linearity stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and prepare 3 portions in the same way.

[0096] (2) Determination: An Agilent 1260 high performance liquid chromatograph equipped with a DAD detector was used. The blank solution and linear test solution were injected according to the chromatographic conditions of Example 1, and the chromatograms were recorded. The linear regression equation was calculated with the average peak area as the x-axis and the average concentration as the y-axis. The linear determination data of the content are shown in Table 15 below, and the linear determination data of related substances are shown in Table 16.

[0097] Table 15 Data on linear determination of content

[0098] Table 16 Data on linearity determination of related substances

[0099] (3) F-test: The F-test is used to evaluate whether there is a significant difference in precision between linear pairs.

[0100] Calculation steps: ① Select a significance level α = 0.05; ② Obtain the standard deviation S of peak area / concentration from 3 measurements, and determine Smax and Smin (Smax>Smin); ③ Calculate the F value, F = S max 2 / S min 2 ; ④ Calculate the degrees of freedom (n2-1, n1-1) = (4, 4), n2 = n1 = 5; ⑤ Consult the one-sided test for F0≤6.39 when α=0.05, degrees of freedom are (4, 4).

[0101] The results of the F-value test for the content are shown in Table 17 below, and the results of the F-value test for related substances are shown in Table 18 below.

[0102] Table 17 Results of F-value Test for Content

[0103] Table 18 Results of F-value tests for related substances

[0104] (4) Results: Content linearity: In the range of 0.4060 mg / ml to 0.6157 mg / ml (equivalent to 80% to 120% of the test sample), the linear correlation coefficient R was [missing value]. 2=0.996, slope 0.00004, residual sum of squares 0.0001, F-value 2.21, ratio of absolute intercept to concentration corresponding to permissible limit 4.42%; related substances linearity: o-dichlorobenzyl in the range of 0.2485 μg / ml~7.4536 μg / ml (equivalent to 0.05%~1.50% of the test sample), linear correlation coefficient R 2 =1.000, slope 0.0381, residual sum of squares 0.01414, F-value 3.78, the ratio of the absolute value of the intercept to the concentration corresponding to the permissible limit 0.14%; o-xylene in the range of 0.2484 μg / ml~7.4532 μg / ml (equivalent to 0.05%~1.50% of the test sample) has a linear correlation coefficient R. 2 =1.000, slope 0.0663, residual sum of squares 0.00155, F-value 2.13, the ratio of the absolute value of the intercept to the concentration corresponding to the permissible limit 1.33%; o-methylbenzyl chloride in the range of 0.2533 μg / ml~7.5989 μg / ml (equivalent to 0.05%~1.50% of the test sample), linear correlation coefficient R 2 =1.000, slope is 0.0366, residual sum of squares is 0.01454, F value is 1.85, and the ratio of the absolute value of the intercept to the concentration corresponding to the allowable limit is 0.04%; all meet the requirements.

[0105] Example 6 Accuracy Test 1. Content accuracy: (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0106] Reference solution: Weigh 10.159 mg of o-methylbenzyl chloride reference standard accurately, place it in a 20 ml volumetric flask, dilute to the mark with solvent and shake well.

[0107] Content accuracy test solution 1: Accurately weigh 8.214 mg, 8.200 mg, and 8.193 mg of o-methylbenzyl chloride reference standard, place them in 20 ml volumetric flasks respectively, dissolve and dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0108] Content accuracy test solution 2: Weigh 10.386 mg, 10.409 mg, and 10.406 mg of o-methylbenzyl chloride reference standard accurately, place them in 20 ml volumetric flasks respectively, dissolve and dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0109] Content accuracy test solution 3: Weigh 12.476 mg, 12.435 mg, and 12.405 mg of o-methylbenzyl chloride reference standard accurately, place them in 20 ml volumetric flasks respectively, dissolve and dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0110] (2) Determination: Using an Agilent 1260 high performance liquid chromatograph equipped with a DAD detector, the reference solution, blank solution and accuracy test solution were injected according to the chromatographic conditions of Example 1, and the chromatograms were recorded. Content recovery rate (%) =

[0111] Note: A 主 The peak area of ​​the main component in the test solution; A 总 It is the sum of the peak areas in the test solution.

[0112] The test data for the accuracy of o-methylbenzyl chloride content are shown in Table 19 below.

[0113] Table 19 Test data on the accuracy of o-methylbenzyl chloride content

[0114] (3) Results: The recovery rate of the content accuracy test solution was in the range of 98.57% to 98.60%, and the relative standard deviation of the recovery rate was 0.01%.

[0115] 2. Accuracy of unknown impurities: (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0116] Reference solution: Accurately weigh 5.131 mg of o-methylbenzyl chloride reference standard, place it in a 5 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, accurately measure 0.5 ml of the above solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0117] Unknown Impurity Accuracy Test Solution 1: Accurately weigh 5.109 mg, 5.111 mg, and 5.124 mg of o-methylbenzyl chloride reference standards, respectively, and place them in 5 ml volumetric flasks. Dissolve and dilute to the mark with solvent, and shake well. Accurately transfer 1.0 ml of the above solution to a 20 ml volumetric flask, add solvent to dilute to the mark, and mix well. Take 0.05 ml of the above solution and place it in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare three parallel solutions.

[0118] Unknown Impurity Accuracy Test Solution 2: Accurately weigh 5.109 mg, 5.111 mg, and 5.124 mg of o-methylbenzyl chloride reference standards, respectively, and place them in 5 ml volumetric flasks. Dissolve and dilute to the mark with solvent, and shake well. Accurately transfer 1.0 ml of the above solution to a 20 ml volumetric flask, add solvent to dilute to the mark, and mix well. Take 1.0 ml of the above solution and transfer it to a 20 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 3 parallel solutions.

[0119] Unknown impurity accuracy test solution 3: Accurately weigh 5.137 mg, 5.146 mg, and 5.171 mg of o-methylbenzyl chloride reference standards, respectively, and place them in 5 ml volumetric flasks. Dissolve and dilute to the mark with solvent, and shake well. Accurately transfer 1.0 ml of the above solution to a 20 ml volumetric flask, add solvent to dilute to the mark, and mix well. Take 2.0 ml of the above solution and place it in a 20 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare 3 parallel solutions.

[0120] Unknown Impurity Accuracy Test Solution 4: Accurately weigh 5.160 mg, 5.171 mg, and 5.147 mg of o-methylbenzyl chloride reference standards, respectively, and place them in 5 ml volumetric flasks. Dissolve and dilute to the mark with solvent, and shake well. Accurately transfer 1.0 ml of the above solution to a 20 ml volumetric flask, add solvent to dilute to the mark, and mix well. Take 2.4 ml of the above solution and place it in a 20 ml volumetric flask, add solvent to dilute to the mark, and shake well. Prepare three parallel solutions.

[0121] (2) Determination: Using an Agilent 1260 high performance liquid chromatograph equipped with a DAD detector, the reference solution, blank solution and accuracy test solution were injected according to the chromatographic conditions of Example 1, and the chromatograms were recorded. Unknown impurity recovery rate (%) =

[0122] Remark: →Test the peak area of ​​o-methylbenzyl chloride in the solution spectrum; →The average peak area of ​​o-methylbenzyl chloride in the spectrum of the reference solution; →Peak area of ​​o-methylbenzyl chloride in the solvent spectrum; →Concentration of the reference solution, µg / ml; →Test solution concentration, µg / ml.

[0123] The test data for the accuracy of unknown impurities are shown in Table 20 below.

[0124] Table 20 Test data for accuracy of unknown impurities

[0125] (3) Results: The recovery rate of the unknown impurity accuracy test solution ranged from 96.30% to 117.40%, and the relative standard deviation of the recovery rate was 5.71%.

[0126] 3. Accuracy of known impurities: (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0127] Blank solution for test sample: Weigh 10.006 mg of o-methylbenzyl chloride accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0128] Reference stock solutions: Weigh approximately 5 mg of o-dichlorobenzyl reference standard in 10 portions, approximately 5 mg of o-xylene reference standard in 10 portions, and approximately 5 mg of o-methylbenzyl chloride reference standard in 3 portions. Accurately weigh each portion and place them in a 5 ml volumetric flask. Add solvent to dissolve and dilute to the mark and mix well. These are used as the stock solutions for each reference standard. One portion is used to prepare the reference solution, and nine portions are used to prepare the accuracy test solution.

[0129] Impurity mixed stock solution: Accurately measure 1.0 ml each of o-dichlorobenzyl and o-xylene reference stock solutions (o-dichlorobenzyl-stock2~10, o-xylene-stock2~10), place them in a 20 ml volumetric flask, add solvent to make up to the mark, and mix well (prepare 9 portions in the same way, and label them as IMP-Stock1-9 respectively).

[0130] Reference solutions: Accurately measure 0.5 ml each of o-dichlorobenzyl and o-xylene reference stock solutions (o-dichlorobenzyl-stock1, o-xylene-stock1), place them in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0131] System suitability solution: Weigh 10.449 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve it, accurately measure 2.0 ml of impurity mixed stock solution (IMP-Stock4), add solvent to dilute to the mark, and shake well.

[0132] Accuracy test solution 1 for known impurities: Accurately measure 0.5 ml each of the stock solutions of o-dichlorobenzyl, o-xylene, and o-methylbenzyl chloride (o-dichlorobenzyl-stock2~4, o-xylene-stock2~4, o-methylbenzyl chloride-stock1-3) and place them in a 100 ml volumetric flask. Add solvent to make up to the mark and mix well. Accurately measure 0.5 ml of the solution and place it in a 10 ml volumetric flask. Add solvent to dilute to the mark and shake well. Prepare 3 aliquots in the same way.

[0133] Accurate test solution 2 for known impurities: Weigh 10.754 mg, 10.710 mg, and 10.754 mg of o-methylbenzyl chloride test sample accurately, place them in 20 ml volumetric flasks respectively, dissolve them in solvent, add 1.0 ml of impurity mixed stock solution (IMP-Stock 1-3) to each flask, dilute to the mark with solvent, and shake well. Prepare 3 flasks using the same method.

[0134] Accuracy test solution 3 for known impurities: Weigh 10.449 mg, 10.406 mg, and 10.463 mg of o-methylbenzyl chloride test sample accurately, place them in 20 ml volumetric flasks respectively, dissolve them in solvent, add 2.0 ml of impurity mixed stock solution (IMP-Stock 4-6) to each flask, dilute to the mark with solvent, and shake well. Prepare 3 aliquots using the same method.

[0135] Accurate test solution 4 for known impurities: Weigh 10.797 mg, 10.827 mg, and 10.798 mg of o-methylbenzyl chloride test sample accurately, place them in 20 ml volumetric flasks respectively, dissolve them in solvent, add 2.4 ml of impurity mixed stock solution (IMP-Stock 7-9) to each flask, dilute to the mark with solvent, and shake well. Prepare 3 aliquots using the same method.

[0136] (2) Determination: Using an Agilent 1260 high performance liquid chromatograph equipped with a DAD detector, and following the chromatographic conditions of Example 1, the system suitability solution, reference solution, blank solution, test sample blank solution, and accuracy test solution were injected and the chromatograms were recorded.

[0137] Recovery rate of each known impurity (%) =

[0138] Remark: →Test the peak area of ​​known impurities in the solution spectrum; →The average peak area of ​​known impurities in the spectrum of the reference solution; →Peak areas of known impurities in the spectrum of the test solution; →Peak areas of known impurities in the solvent spectrum; →Weighing of known impurities added to the test solution, in mg; →Weigh the o-methylbenzyl chloride in the test solution, in mg; →Concentration of the reference solution, µg / ml; →Test solution concentration, µg / ml. The accuracy test data for o-dichlorobenzyl are shown in Table 21 below.

[0139] Table 21 Accuracy Test Data for o-Dichlorobenzyl

[0140] The accuracy test data for o-xylene are shown in Table 22 below.

[0141] Table 22 Accuracy Test Data for o-xylene

[0142] (3) Results: The recovery rates of o-dichlorobenzyl impurity accuracy test solutions 1-4 were in the range of 95.55% to 105.28%, and the relative standard deviation of the recovery rate was 2.14%; the recovery rates of o-xylene impurity accuracy test solutions 1-4 were in the range of 86.96% to 106.88%, and the relative standard deviation of the recovery rate was 7.16%; both met the requirements.

[0143] Based on the above tests of precision, linearity, and accuracy, it can be determined that: Content: o-methylbenzyl chloride is within the range of 0.4060 mg / ml to 0.6157 mg / ml (equivalent to 80% to 120% of the test sample). Related substances: o-dichlorobenzyl is within the range of 0.2485 μg / ml to 7.4536 μg / ml (equivalent to 0.05% to 1.50% of the test sample), o-xylene is within the range of 0.2484 μg / ml to 7.4532 μg / ml (equivalent to 0.05% to 1.50% of the test sample), and o-methylbenzyl chloride is within the range of 0.2533 μg / ml to 7.5989 μg / ml (equivalent to 0.05% to 1.50% of the test sample). The analytical method is applicable in all these cases, and the results obtained are accurate and reliable.

[0144] Example 7 Solution stability test (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0145] Reference stock solution: Accurately weigh the corresponding amount of reference standard according to Table 23, place it in the corresponding volumetric bottle, add solvent to dissolve and dilute to the mark, and shake well.

[0146] Table 23 Weighing and concentration data of the reference standard stock solution

[0147] Impurity mixed stock solution: Accurately measure 1.0 ml of o-dichlorobenzyl and o-xylene reference stock solution, place it in a 20 ml volumetric flask, add solvent to dilute to the mark, and mix well.

[0148] System suitability solution: Weigh 10.365 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve it, accurately measure 2.0 ml of impurity mixed stock solution, dilute to the mark with solvent, and shake well.

[0149] Reference solutions: Accurately measure 0.5 ml each of o-methylbenzyl chloride, o-dichlorobenzyl chloride, and o-xylene reference stock solutions, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0150] Solution stability test solution: Weigh 10.190 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve it, accurately measure 2.0 ml of impurity mixed stock solution, add solvent to dilute to the mark, shake well, and test the stability of the vial and volumetric flask.

[0151] Test solution: Weigh 10.259 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and test the stability of the vial and volumetric flask.

[0152] (2) Testing: Using an Agilent 1260 high performance liquid chromatograph equipped with a DAD detector, and following the chromatographic conditions of Example 1, at appropriate time points, the above system suitability solution, reference solution, blank solution, test solution, and solution stability test solution were injected and the chromatograms were recorded.

[0153] The stability test solution data for the vials are shown in Table 24 below.

[0154] Table 24 Stability Test Solution Measurement Data - Injection Vial

[0155] The stability test data of the volumetric flask solution are shown in Table 25 below.

[0156] Table 25 Stability Test Solution Measurement Data - Volumetric Flasks

[0157] The test solution measurement data of the vials are shown in Table 26 below.

[0158] Table 26 Test Solution Determination Data - Injection Vial

[0159] The test data for the volumetric flask sample solution are shown in Table 27 below.

[0160] Table 27 Test Sample Solution Determination Data - Volumetric Flask

[0161] (3) Results: Under the solution stability test, the maximum range of target impurity content in the test solution placed in the vial within 82 hours was 0.03%, the maximum range of unknown single impurity content was 0.01%, and the maximum range of total impurity content was 0.04%; the maximum range of target impurity content in the stability test solution within 82 hours was 0.10%, the maximum range of other single impurity content was 0.01%, and the maximum range of total impurity content was 0.09%; the maximum range of target impurity content in the test solution placed in the volumetric flask at room temperature within 84 hours was 0.01%, the maximum range of unknown single impurity content was 0.02%, and the maximum range of total impurity content was 0.02%; the maximum range of target impurity content in the stability test solution within 84 hours was 0.04%, the maximum range of other single impurity content was 0.01%, and the maximum range of total impurity content was 0.05%. Therefore, the test solution placed in the volumetric flask at room temperature was stable within 84 hours, and the test solution placed in the vial was stable within 82 hours.

[0162] Example 8 Durability Test (1) Solution preparation Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0163] Reference stock solution: Accurately weigh the corresponding amount of reference standard according to Table 28, place it in the corresponding volumetric bottle, add solvent to dissolve and dilute to the mark, and shake well.

[0164] Table 28 Weighing and Concentration Data of Reference Standard Stock Solution

[0165] Impurity mixed stock solution: Accurately measure 1.0 ml of o-dichlorobenzyl and o-xylene reference stock solution, place it in a 20 ml volumetric flask, add solvent to make up to the mark, and mix well.

[0166] System suitability solution: Weigh 10.365 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, dissolve it in solvent, add 2.0 ml of impurity mixed stock solution, and dilute to the mark with solvent. Shake well.

[0167] Reference solutions: Accurately measure 0.5 ml each of o-methylbenzyl chloride, o-dichlorobenzyl chloride, and o-xylene reference stock solutions, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0168] Durability test solution: Weigh 10.190 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, dissolve it in solvent, add 2.0 ml of impurity mixed stock solution, and dilute to the mark with solvent. Shake well.

[0169] (2) Testing: An Agilent 1260 high-performance liquid chromatograph equipped with a DAD detector was used. Under the original chromatographic conditions of Example 1, column temperature ±2℃ (38℃, 42℃), flow rate ±5% (0.95ml / min, 1.05ml / min), and initial mobile phase methanol ±5% (0.1% phosphoric acid water-methanol (45:55), 0.1% phosphoric acid water-methanol (35:65)), system suitability solution, reference solution, blank solution, and robustness test solution were injected and the chromatograms were recorded. The robustness test data are shown in Table 29 below.

[0170] Table 29 Durability Test Data

[0171] (3) Results: Under the original conditions and various durability conditions, the maximum range of the target impurity content was 0.06%, the maximum range of other single impurity content was 0.04%, and the maximum range of total impurity content was 0.07%. All durability conditions met the requirements.

[0172] Example 9 Test Method Experiment (1) Solution preparation: Blank solution (solvent): Acetonitrile (5A molecular sieve for water removal).

[0173] Reference standard stock solution: Accurately weigh the corresponding amount of reference standard according to Table 30, place it in the corresponding volumetric bottle, add solvent to dissolve and dilute to the mark, and shake well.

[0174] Table 30 Weighing and Concentration Data of Reference Standard Stock Solution

[0175] Reference solutions: Accurately measure 0.5 ml each of o-methylbenzyl chloride, o-dichlorobenzyl chloride, and o-xylene reference stock solutions, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0176] Test solution: Weigh 10.46 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0177] (2) Chromatographic conditions: Instrument: Shimadzu LC-20AT high performance liquid chromatograph with PDA detector; Column: Agilent ZORBAX SB-C18 4.6×250mm 5μm; Detection wavelength: 220nm; Mobile phase A: 0.05% aqueous phosphoric acid solution; Mobile phase B: Methanol; Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL; The gradient elution procedure is the same as in Table 3 of Example 1.

[0178] (3) Determination: Accurately measure the blank solution, reference solution and test solution and inject them into the liquid chromatograph. Record the chromatogram. The blank solution showed no significant interference at the main peak of o-methylbenzyl chloride and the peaks of o-dichlorobenzyl and o-xylene. The test results of the test sample are shown in Table 31 below.

[0179] Table 31 Test Results of the Sample

[0180] Example 10 Test Method Experiment (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0181] Reference standard stock solution: Accurately weigh the corresponding amount of reference standard according to Table 32, place it in the corresponding volumetric bottle, add solvent to dissolve and dilute to the mark, and shake well.

[0182] Table 32 Weighing and Concentration Data of Reference Standard Stock Solution

[0183] Reference solutions: Accurately measure 0.5 ml each of o-methylbenzyl chloride, o-dichlorobenzyl chloride, and o-xylene reference stock solutions, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0184] Test solution: Weigh 10.69 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0185] (2) Chromatographic conditions: Instrument: Agilent 1260 high performance liquid chromatograph with DAD detector; Chromatographic column: YMC ODS-AQ 4.6×250mm 5μm; Detection wavelength: 220nm; Mobile phase A: 0.2% aqueous phosphoric acid solution; Mobile phase B: Methanol; Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL; The gradient elution procedure is the same as in Table 3 of Example 1.

[0186] (3) Determination: Accurately measure the blank solution, reference solution and test solution and inject them into the liquid chromatograph. Record the chromatogram. The blank solution showed no significant interference at the main peak of o-methylbenzyl chloride and the peaks of o-dichlorobenzyl and o-xylene. The test results of the test sample are shown in Table 33 below.

[0187] Table 33 Test Results of the Sample

[0188] Example 11 Sample Testing Test (1) Solution preparation: Blank solution (solvent): acetonitrile (anhydrous sodium sulfate to remove water).

[0189] Reference stock solution: Accurately weigh the corresponding amount of reference standard according to Table 34, place it in the corresponding volumetric bottle, add solvent to dissolve and dilute to the mark, and shake well.

[0190] Table 34 Weighing and Concentration Data of Reference Standard Stock Solution

[0191] Reference solutions: Accurately measure 0.5 ml each of o-methylbenzyl chloride, o-dichlorobenzyl chloride, and o-xylene reference stock solutions, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and mix well.

[0192] Test solution: Weigh approximately 10.32 mg of o-methylbenzyl chloride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0193] (2) Chromatographic conditions: Instrument: Shimadzu LC-20AT high performance liquid chromatograph with UV detector; Column: Agilent ZORBAX SB-C18 4.6×250mm 5μm; Detection wavelength: 220nm; Mobile phase A: 0.1% aqueous phosphoric acid solution; Mobile phase B: Methanol; Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL; The gradient elution procedure is the same as in Table 3 of Example 1.

[0194] (3) Determination: Accurately measure blank solution, reference solution and test solution and inject them into the liquid chromatograph. Record the chromatograms. The blank solution showed no significant interference at the main peak of o-methylbenzyl chloride and the peaks of o-dichlorobenzyl and o-xylene. The test results of the test sample are shown in Table 35 below. The HPLC chromatogram of the test sample solution in Example 11 is shown below. Figure 7 As shown.

[0195] Table 35 Test Results of the Sample

Claims

1. An HPLC quantitative detection method for o-methylbenzyl chloride and its impurities, characterized in that, Prepare blank solution, test solution and reference solution. Inject the blank solution, test solution and reference solution into the liquid chromatograph and record the chromatograms. Calculate the content of o-methylbenzyl chloride in the test solution by peak area normalization method. Calculate the content of impurities o-dichlorobenzyl and / or o-xylene in the test solution by peak area using external standard method. Calculate the content of other impurities in the test solution by main component self-comparison method. The structural formula of o-methylbenzyl chloride is as follows: ； The structural formula of o-dichlorobenzyl is as follows: ； The structural formula of o-xylene is as follows: ； The chromatographic conditions in the HPLC method include the mobile phase; the mobile phase is an aqueous solution of phosphoric acid as mobile phase A and methanol as mobile phase B. The chromatographic conditions also include a gradient elution program, which is as follows: 0-2 minutes, mobile phase A accounts for 35-45% of the total mobile phase volume, and mobile phase B accounts for 55-65% of the total mobile phase volume; 2-15 minutes, mobile phase A accounts for 25-35% of the total mobile phase volume, and mobile phase B accounts for 65-75% of the total mobile phase volume; 15-20 minutes, mobile phase A accounts for 25-35% of the total mobile phase volume, and mobile phase B accounts for 65-75% of the total mobile phase volume; 20-25 minutes, mobile phase A accounts for 35-45% of the total mobile phase volume, and mobile phase B accounts for 55-65% of the total mobile phase volume; 25-30 minutes, mobile phase A accounts for 35-45% of the total mobile phase volume, and mobile phase B accounts for 55-65% of the total mobile phase volume.

2. The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities according to claim 1, characterized in that, The test solution is prepared by weighing an o-methylbenzyl chloride sample, dissolving and diluting it with a solvent to prepare the test solution; the concentration of o-methylbenzyl chloride in the test solution is 0.4-0.6 mg / mL.

3. The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities according to claim 1, characterized in that, The reference solution is prepared by diluting the stock solution of any single component or mixture of o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride with a solvent to prepare the reference solution; the concentration of any single component in the solution of any single component or mixture of o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride is 0.25-7.5 μg / mL.

4. The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities according to claim 3, characterized in that, The preparation method of the stock solution is as follows: weigh o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride reference standards respectively, dissolve and dilute them with solvent to prepare a stock solution containing any single component or mixture of o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride; the concentration of any single component in the solution of any single component or mixture of o-dichlorobenzyl, o-xylene and o-methylbenzyl chloride in the stock solution is 2.5-1200 μg / mL.

5. The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities according to any one of claims 2-4, characterized in that, The solvent is acetonitrile.

6. The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities according to claim 1, characterized in that, The mobile phase A is a 0.05 wt.% to 0.2 wt.% aqueous solution of phosphoric acid.

7. The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities according to claim 1, characterized in that, The parameters and conditions for the liquid chromatography include: the chromatographic column is a C18 column with octadecylsilane-bonded silica gel as the packing material, and the column temperature is 38-42℃.

8. The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities according to claim 7, characterized in that, The parameters for the liquid chromatography include: the detector is a DAD / PDA detector or an ultraviolet detector, and the detection wavelength is 218-222 nm.

9. The HPLC quantitative detection method for o-methylbenzyl chloride and its impurities according to claim 1, characterized in that, The total flow rate of mobile phase A and mobile phase B is 0.95-1.05 mL / min.

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