Analytical Method for Genotoxic Impurity of 2-Chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in Etoricoxib

Abstract

The invention discloses an analysis method of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate genotoxic impurities in etoricoxib, adopting an HPLC analysis method, wherein the following are adopted: the instrument is a high performance liquid chromatograph with an ultraviolet detector; mobile phase: A: phosphate and ion pair reagent buffer, B: methanol; gradient elution; detection wavelength: 310nm; column temperature: 30-40°C; injection volume: 10μl; solvent is: mobile phase A: B=30:70; test solution: concentration is 10mg / ml; reference solution: concentration is 0.125μg / ml; determination process: blank solution, reference solution and test solution are respectively sampled and analyzed in sequence; content is calculated by peak area according to an external standard method. The analysis method of the invention can be analyzed by conventional liquid chromatography method when the limit of genotoxic impurities in the drug is low, i.e., the determination is accurate, thereby reducing the detection cost.

Description

Technical Field

[0001] The invention belongs to a drug analysis method, and in particular relates to an analysis method for genotoxic impurities of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib. Background Art

[0002] "Genotoxic impurities" (also known as genotoxic impurities, GTI) refer to compounds that directly or indirectly damage cell DNA, produce gene mutations or in vivo mutagenesis, and have the potential or tendency to cause cancer. Its main sources are starting materials, intermediates, reagents and reaction by-products in the synthesis of raw materials. In addition, drugs may also produce genotoxic impurities due to degradation during synthesis, storage or preparation. Because of their extremely strong toxicity, they can cause damage to human genetic material at very low concentrations, leading to gene mutations and possibly promoting the occurrence of tumors, posing a strong threat to the safety of medication. Therefore, it is very necessary to establish a convenient and efficient analysis method.

[0003] The commonly used methods for detecting genotoxic impurities include: high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS), etc.

[0004] 2-Chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate is used to prepare etoricoxib. It is a heat shock stable and non-hygroscopic solid. For etoricoxib, it is very important to accurately detect the amount of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate genotoxic impurities in it for drug safety. However, because the genotoxic impurity limit of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib is low and must not exceed 12.5ppm, for such a low detection limit, most companies will choose to use more sensitive GC-MS or LC-MS for detection, but GC-MS and LC-MS are very expensive, which also brings a considerable burden to companies. In order to save costs, people hope to find a better low-cost but high-precision detection method. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for analyzing the genotoxic impurity of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib with low cost and high detection accuracy.

[0006] The present invention is achieved through the following technical solutions:

[0007] The analytical method for the genotoxic impurity of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib is a high performance liquid chromatography HPLC analytical method, wherein:

[0008] The instruments were: high performance liquid chromatograph with UV detector;

[0009] Chromatographic column: Aeger Venusil XBP C18 (4.6 mm × 250 mm, 5 μm);

[0010] Mobile phase: A: phosphate and ion pair reagent buffer (weigh 2.72 g potassium dihydrogen phosphate, add 2.16 g sodium octane sulfonate, add 1000 ml water to dissolve, and adjust the pH to 3.5 with 10% dilute phosphoric acid solution), B: methanol;

[0011] Gradient elution:

[0012]

[0013] Flow rate: 1.0ml / min;

[0014] Detection wavelength: 310nm; at this wavelength, the absorption intensity of other impurities is low and the peak is small, while 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate has strong absorption and a large peak area, which can better distinguish it from other impurities;

[0015] Column temperature: 20-40°C;

[0016] Injection volume: 10 μl;

[0017] The solvent is: mobile phase A:B=30:70;

[0018] Test solution: Accurately weigh 100 mg of etoricoxib API into a 10 ml volumetric flask, dissolve in solvent and dilute to 10 ml mark to obtain a test solution with a concentration of 10 mg / ml;

[0019] Reference substance solution: accurately weigh 50 mg of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate reference substance into a 100 ml volumetric flask, add solvent to dissolve and dilute to 100 ml, then continue to dilute to a concentration of 0.125 μg / ml, shake well, and obtain the reference substance solution;

[0020] Determination process: Take blank solution (solvent), reference solution and test solution and inject them in turn, the injection volume is 10μl, and record the chromatogram;

[0021] The content of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in the test sample was calculated by the peak area using the external standard method.

[0022] Preferably, the calculation formula for calculating the content of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in the test sample by peak area according to the external standard method is:

[0023]

[0024] Where A SAP : The peak area of ​​2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) in the test solution;

[0025] A STD : Peak area of ​​2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) of reference solution;

[0026] C STD : Concentration of reference solution (mg / mL);

[0027] C SAP : Concentration of test solution (mg / mL).

[0028] As a preferred method, the result judgment is: if there is a 2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) (ET-SM2) peak in the chromatogram of the test solution, it should not exceed 12.5ppm according to the external standard method.

[0029] The reason why most companies need to use more sensitive GC-MS or LC-MS to detect impurities with lower limits is that, firstly, the limits themselves are low and the amount is small, so it is not easy to detect. Secondly, impurities will affect each other and affect the determination. Therefore, it is generally necessary to use more sensitive GC-MS or LC-MS for detection. However, the cost of GC-MS and LC-MS is very high, and long-term detection will bring a considerable burden to the company. For this reason, the inventor has been committed to how to reduce this cost.

[0030] The inventors found in their research that the separation between the genotoxic impurity of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in torcoxib is poor and easily interfered by other impurities; and the impurity limit is low, and a method with high sensitivity needs to be established for effective detection. Therefore, the optimal gradient elution method was first explored to separate other impurities from 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate well, and the optimal wavelength was selected at the same time. Finally, the wavelength of 310nm was used. At this wavelength, the absorption intensity of other impurities is low and the peak is small, while 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate has strong absorption and a large peak area, so as to better distinguish it from other impurities. On the basis of good separation, a high concentration of test solution was used to increase the amount of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in the test solution as much as possible, so that more 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate can enter the chromatograph, thereby improving its detection capability. Finally, the above technical solution was obtained, which can accurately detect genotoxic impurities with very low control limits using conventional liquid chromatography methods.

[0031] The beneficial effects of the present invention are:

[0032] The method for analyzing genotoxic impurities of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib of the present invention has ingenious steps. The separation degree is firstly improved, and then the detection capability is improved. The two methods are coordinated to achieve the excellent effect that the conventional and universal liquid chromatography HPLC method can be used for analysis when the limit of genotoxic impurities in drugs is low, that is, the impurity content of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib can be accurately determined. The method perfectly replaces the LCMS detection method with higher sensitivity that most enterprises would choose to use, greatly reducing the detection cost of enterprises and reducing the burden on enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] For ease of explanation, the present invention is described in detail with reference to the following specific embodiments and accompanying drawings.

[0034] Figure 1 The ultraviolet scanning diagram of the determined wavelength of the method of the present invention, wherein the abscissa represents the wavelength in nm, and the ordinate represents the absorption value in A;

[0035] Figure 2 This is the ET-SM2 positioning chromatogram under the conditions of method 1 in the establishment of chromatographic conditions of the method of the present invention, in which the abscissa represents time in min, and the ordinate represents the chromatographic signal value in mAU;

[0036] Figure 3The chromatogram of each impurity mixed solution under the conditions of method 1 in establishing the chromatographic conditions of the method of the present invention, wherein the abscissa represents time in min, and the ordinate represents the chromatographic signal value in mAU;

[0037] Figure 4 This is the ET-SM2 positioning chromatogram under the conditions of method 2 in establishing the chromatographic conditions of the method of the present invention, in which the abscissa represents time in min, and the ordinate represents the chromatographic signal value in mAU;

[0038] Figure 5 The chromatograms of the impurity mixed solutions under the conditions of method 2 in establishing the chromatographic conditions of the method of the present invention are shown in FIG. 1 , wherein the horizontal axis represents time in min, and the vertical axis represents the chromatographic signal value in mAU;

[0039] Figure 6 This is item three in the method validation of the method of the present invention: c. Linear regression equation diagram in linearity and range, in which the horizontal axis represents concentration in ug / ml, and the vertical axis represents peak area in μV*sec. DETAILED DESCRIPTION

[0040] The analytical method for the genotoxic impurity of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib is a high performance liquid chromatography HPLC analytical method, wherein:

[0041] The instruments were: high performance liquid chromatograph with UV detector;

[0042] Chromatographic column: Aeger Venusil XBP C18 (4.6 mm × 250 mm, 5 μm);

[0043] Mobile phase: A: phosphate and ion pair reagent buffer (weigh 2.72 g potassium dihydrogen phosphate, add 2.16 g sodium octane sulfonate, add 1000 ml water to dissolve, and adjust the pH to 3.5 with 10% dilute phosphoric acid solution), B: methanol;

[0044] Gradient elution:

[0045]

[0046] Flow rate: 1.0ml / min;

[0047] Detection wavelength: 310nm; at this wavelength, the absorption intensity of other impurities is low and the peak is small, while 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate has strong absorption and a large peak area, which can better distinguish it from other impurities;

[0048] Column temperature: 35°C;

[0049] Injection volume: 10 μl;

[0050] The solvent is: mobile phase A:B=30:70;

[0051] Test solution: Accurately weigh 100 mg of etoricoxib API into a 10 ml volumetric flask, dissolve in solvent and dilute to 10 ml mark to obtain a test solution with a concentration of 10 mg / ml;

[0052] Reference substance solution: accurately weigh 50 mg of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate reference substance into a 100 ml volumetric flask, add solvent to dissolve and dilute to 100 ml, then continue to dilute to a concentration of 0.125 μg / ml, shake well, and obtain the reference substance solution;

[0053] Determination process: Take blank solution (solvent), reference solution and test solution and inject them in turn, the injection volume is 10μl, and record the chromatogram;

[0054] The content of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in the test sample was calculated by the peak area according to the external standard method. The calculation formula is:

[0055]

[0056] Where A SAP : The peak area of ​​2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) in the test solution;

[0057] A STD : Peak area of ​​2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) of reference solution;

[0058] C STD : Concentration of reference solution (mg / mL);

[0059] C SAP : Concentration of test solution (mg / mL).

[0060] Result judgment: If there is a 2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) peak in the chromatogram of the test solution, it should not exceed 12.5ppm when calculated by the external standard method.

[0061] To simplify the description, in this embodiment, 2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) is referred to as ET-SM2.

[0062] Among them, during the method establishment and optimization process:

[0063] 1. Determination of detection wavelength:

[0064] Take ET-SM2 and dissolve it in acetonitrile and dilute it to make a solution containing 10μg per 1ml. Perform UV scanning in the wavelength range of 200nm to 400nm. The scanned UV image of ET-SM2 is as shown in the attached figure. Figure 1 As shown in the figure, ET-SM2 has a large absorption at a wavelength of 310 nm, so the detection wavelength is set to 310 nm.

[0065] 2. Determination of mobile phase:

[0066] Potassium dihydrogen phosphate buffer (pH 3.5) was used for the experiment. Since ET-SM2 has a strong polarity and an early peak time, its retention behavior on the chromatographic column was improved by adding an ion pair reagent. Finally, the mobile phase was determined to be a pH 3.5 phosphate buffer (2.72 g potassium dihydrogen phosphate and 2.16 g sodium octane sulfonate were weighed, dissolved in 1000 ml water, and the pH was adjusted to 3.5 with 10% phosphoric acid); the organic phase was methanol.

[0067] 3. Establishment and optimization of chromatographic conditions:

[0068] The isocratic method was used, and the main peak shape was poor, so a gradient elution method was proposed for detection. The chromatographic conditions and results are shown in the following table:

[0069] Method establishment and optimization results table (method one)

[0070]

[0071] Method establishment and optimization results table (method 2)

[0072]

[0073] 4. Determination of test sample concentration:

[0074] The test sample concentration is 1 mg / mL, the quantification limit is 0.30 ng, and the injection volume is 10 μL. The minimum detection limit of this method is 30 ppm. If the test sample concentration is increased to 10 mg / mL, the quantification limit and injection volume remain unchanged, the minimum detection limit is 3 ppm, which can meet the limit requirement of 12.5 ppm. Therefore, the test sample concentration is determined to be 10 mg / mL.

[0075] Method validation

[0076] The analytical method was validated according to the 2020 edition of the Chinese Pharmacopoeia, Part IV, "Guidelines for Validation of Analytical Methods", and all validation items met the requirements of the Pharmacopoeia. The main contents are shown in Table 1 below:

[0077] Table 1 Summary of validation of ET-SM2 method in etoricoxib

[0078]

[0079] The specific contents of the method validation are as follows:

[0080] Project 1: Exclusivity

[0081] a. Blank solvent interference

[0082] The interference of blank solvent on ET-SM2 test was investigated.

[0083] ①Blank solvent: mobile phase A:B=30:70;

[0084] ② Reference solution: take about 10 mg of ET-SM2 reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 mL, place it in a 100 mL volumetric flask, add solvent to dilute to the scale, shake well, as the reference solution (1 μg / mL);

[0085] Accurately measure 10 μL of the blank solvent and reference solution mentioned above, inject them into the liquid chromatograph according to the chromatographic conditions determined in this method, and record the chromatogram.

[0086] Conclusion: ET-SM2 elutes at 8.765 min, and the blank solvent has no chromatographic peak at the ET-SM2 elute position, which does not interfere with the detection of ET-SM2.

[0087] b. System suitability

[0088] The separation degree between etoricoxib and its impurities and ET-SM2 was investigated to determine the applicability of the ET-SM2 inspection method.

[0089] ① Impurity mother solution: Take about 10 mg of each of 9 impurities different from ET-SM2, place them in 20 mL volumetric flasks, add solvent to dissolve and dilute to scale, shake well, and use as impurity mother solution (0.5 mg / mL);

[0090] ②ET-SM2 stock solution: Take about 10 mg of ET-SM2 reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 5 mL, place it in a 50 mL volumetric flask, add solvent to dilute to the scale, shake well, as ET-SM2 stock solution (10 μg / mL);

[0091] ③ System suitability solution: Take about 10 mg of etoricoxib, accurately weigh it, put it in a 10 mL volumetric flask, add 1 mL of ET-SM2 stock solution and 20 μL of each impurity mother solution, shake well, and use it as the system suitability solution;

[0092] ④Mixed impurity solution: Take 20 μL of each impurity mother solution, place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and use it as the mixed impurity solution;

[0093] Accurately measure 10 μL of each of the above solutions ③ and ④, inject them into the liquid chromatograph, and use a diode array detector (DAD) to check the peak purity of ET-SM2 and record the chromatogram. The results are shown in Table 2 below.

[0094] Table 2 Method verification system suitability investigation results

[0095]

[0096] Conclusion: In the system suitability test, etoricoxib and other impurities have no chromatographic peaks at the ET-SM2 peak position and do not interfere with the detection of ET-SM2; the separation degrees of ET-SM2 and adjacent impurities are 5.4 and 4.4 respectively; the purity of the main peak of ET-SM2 is greater than 0.9999, and no impurities are detected, indicating that the system applicability of this method is good.

[0097] c. Destruction test

[0098] The effects of degradation products produced under strong alkali, strong acid, oxidation, high temperature and light conditions on the determination of ET-SM2 were investigated. The sample placement conditions and preparation process are shown in Table 3.

[0099] Table 3 Method verification destructive test sample list

[0100]

[0101]

[0102] Take 10 μL of each of the above sample solutions, inject them into the liquid chromatograph according to the chromatographic conditions determined under the method establishment item, and record the chromatogram.

[0103] Conclusion: All the destroyed samples and blank solvents have no chromatographic peaks at the ET-SM2 peak position, indicating that the destruction test under severe conditions does not interfere with the inspection of this product.

[0104] Project 2: ab quantification limit and detection limit

[0105] The sensitivity of the method was examined. When the signal-to-noise ratio of the ET-SM2 peak to the baseline was approximately 10:1, it was the limit of quantification, and when the signal-to-noise ratio was approximately 3:1, it was the limit of detection.

[0106] Take an appropriate amount of ET-SM2 reference substance, accurately weigh it, dilute it step by step to make a series of test solution, accurately measure 10 μL of the test solution of appropriate concentration, inject it into the liquid chromatograph according to the chromatographic conditions determined by the method of the present invention, and record the chromatogram. The determination results are shown in Tables 4 to 6.

[0107] Table 4 Results of the quantitative limit investigation of the ET-SM2 method for the method of the present invention in etoricoxib

[0108]

[0109]

[0110] Table 5 Results of the quantitative limit investigation of the ET-SM2 method for the method of the present invention in etoricoxib

[0111]

[0112] Table 6 Results of the ET-SM2 method validation detection limit investigation in the method of the present invention for etoricoxib

[0113]

[0114] Conclusion: The detection limit concentration of ET-SM2 is 0.00512μg / mL, equivalent to 0.00005% of the test sample concentration, and the signal-to-noise ratio is not less than 3; the quantification limit concentration is 0.01280μg / mL, equivalent to 0.00013% of the test sample concentration, and the signal-to-noise ratio is not less than 10; the RSD% of the peak area of ​​6 consecutive injections of the same solution is 3.3%, and the RSD% of the peak area of ​​6 consecutive solutions is 4.6%, which meets the requirements.

[0115] Item 3: c. Linearity and range

[0116] Through the linearity test of ET-SM2, the linear range of ET-SM2 is obtained.

[0117] ① Reference substance stock solution: Take 10 mg of ET-SM2 reference substance, weigh accurately, place in a 10 mL volumetric bottle, add solvent to dissolve and dilute to scale, shake well. Take 1 mL of the above solution to a 20 mL volumetric bottle, add solvent to dilute to scale, shake well. Take 1 mL of the above solution to a 100 mL volumetric bottle, add solvent to dilute to scale, shake well.

[0118] ②Linear solution 1 (400%): Take the reference substance stock solution as linear solution 1.

[0119] ③Linear solution 2 (200%): Accurately pipette 5 mL of the reference stock solution into a 10 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0120] ④Linear solution 3 (100%): Accurately pipette 5 mL of the reference stock solution into a 20 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0121] ⑤Linear solution 4 (20%): Accurately pipette 1 mL of the reference stock solution into a 20 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0122] ⑥Linear solution 5 (quantitation limit): Accurately pipette 5 mL of linear solution 4 (20%) into a 10 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0123] Accurately measure 10 μL of each of the above solutions ② to ⑥, inject into the liquid chromatograph according to the determined chromatographic conditions, and record the chromatogram. Perform linear regression with concentration as the horizontal axis (X) and peak area as the vertical axis (Y). The results are shown in Table 7, and the regression equation diagram is shown in Figure 6 .

[0124] Table 7 Etoricoxib linear test results of the present invention method

[0125]

[0126] Conclusion: The concentration of ET-SM2 has a good linear relationship with the peak area in the range of 0.01275μg / mL to 0.50998μg / mL.

[0127] Project 4: d. Accuracy and repeatability

[0128] ET-SM2 at the limit of quantitation, 50%, 100%, and 150% concentrations was added to the test solution to investigate the accuracy and repeatability of the test results.

[0129] ① Reference substance stock solution: Weigh 50mg ET-SM2, weigh accurately, place in a 100mL volumetric flask, add solvent to dissolve and dilute to scale, shake well. Then accurately transfer 5mL of the above solution to a 20mL volumetric flask, add solvent to dilute to scale, shake well. Then accurately transfer 1mL of the above solution to a 200mL volumetric flask, add solvent to dilute to scale, shake well to obtain the reference substance stock solution;

[0130] ② Test solution: Weigh 100 mg of etoricoxib test sample, accurately weigh, place in a 10 mL volumetric flask, add solvent to dissolve by ultrasonication and dilute to scale, shake well, and use as the test solution. Prepare two portions in the same way.

[0131] ③ Reference substance solution: Accurately pipette 2 mL of reference substance stock solution into a 10 mL volumetric flask, add solvent to dilute to the scale, and shake well.

[0132] ④ Reference solution 2: Weigh 50mg ET-SM2, weigh accurately, place in a 100mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well. Then accurately transfer 5mL of the above solution to a 20mL volumetric flask, add solvent to dilute to the scale, shake well. Then accurately transfer 1mL of the above solution to a 200mL volumetric flask, add solvent to dilute to the scale, shake well. Then accurately transfer 2mL of the above solution to a 10mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain;

[0133] ⑤ Accuracy solution 1 (quantitative limit): Accurately pipette 1 mL of the reference stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well. Take 2 mL of the above solution into a 10 mL volumetric flask, add 100 mg of etoricoxib test sample, dissolve by ultrasonication with solvent and dilute to the mark, shake well, and the solution is ready. Prepare 3 portions in the same way;

[0134] ⑥ Accuracy solution 2 (50%): Accurately pipette 1 mL of the reference stock solution into a 10 mL volumetric flask, then add 100 mg of etoricoxib test solution, add solvent to dissolve by ultrasonication and dilute to the scale, shake well, and the solution is ready. Prepare 3 portions in the same way;

[0135] ⑦ Accuracy solution 3 (100%): Accurately pipette 2 mL of the reference stock solution into a 10 mL volumetric flask, then add 100 mg of etoricoxib test sample, add solvent to dissolve by ultrasonication and dilute to the scale, shake well, and the solution is ready. Prepare 6 portions in the same way;

[0136] ⑧ Accuracy solution 4 (150%): Accurately pipette 3 mL of the reference stock solution into a 10 mL volumetric flask, then add 100 mg of etoricoxib test solution, add a solvent to dissolve by ultrasonication and dilute to the scale, shake well, and the solution is ready. Prepare 3 portions in the same way;

[0137] Accurately measure 10 μL of each of the above solutions ② to ⑧, inject into the liquid chromatograph according to the determined chromatographic conditions, and record the chromatogram. The results are shown in Tables 8 and 9.

[0138] Table 8 Etoricoxib method accuracy test results

[0139]

[0140] Table 9 Etoricoxib Method of the Invention Repeatability Test Results

[0141]

[0142] Accuracy conclusion: At the limit of quantitation level, the recoveries of the three solutions were all between 95% and 101%; at other concentrations, the recoveries measured were all between 97% and 107%, and the RSD of the recoveries of the nine solutions was 3.2%, indicating that this method has good accuracy.

[0143] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims.

Claims

1. A method for analyzing genotoxic impurities of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib, characterized in that: The high performance liquid chromatography (HPLC) analysis method was used, wherein: The instruments were: high performance liquid chromatograph with UV detector; Chromatographic column: Aeger Venusil XBP C18, specification: 4.6mm×250mm, 5μm; Mobile phase: A: Phosphate and ion pair reagent buffer: weigh 2.72g of potassium dihydrogen phosphate, add 2.16g of sodium octane sulfonate, add 1000ml of water to dissolve, and adjust the pH to 3.5 with 10% dilute phosphoric acid solution; B: methanol; Gradient elution: Flow rate: 1.0ml / min; Detection wavelength: 310nm; Column temperature: 30-40°C; Injection volume: 10 μl; The solvent is: mobile phase A:B=30:70; Test solution: accurately weigh 100 mg of etoricoxib API into a 10 ml volumetric flask, dissolve with solvent and dilute to 10 ml mark to obtain a test solution with a concentration of 10 mg / ml; Reference solution: accurately weigh 50 mg of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate reference substance into a 100 ml volumetric flask, dissolve with solvent and dilute to 100 ml mark, then continue to dilute to a concentration of 0.125 μg / ml, shake well to obtain a reference solution; Determination process: Take blank solution (solvent), reference solution and test solution and inject them in turn, the injection volume is 10μl, and record the chromatogram; The content of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in the test sample was calculated by the peak area using the external standard method.

2. The method for analyzing genotoxic impurities of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in etoricoxib according to claim 1, characterized in that: The calculation formula for calculating the content of 2-chloro-1,3-bis(dimethylamino)trimethylene hexafluorophosphate in the test sample by peak area according to the external standard method is: Where A SAP : The peak area of ​​2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) in the test solution; A STD : Peak area of ​​2-chloro-1,3-(bis(dimethylamino)trimethylene hexafluorophosphate) of reference solution; C STD : Concentration of reference solution (mg / mL); C SAP : Concentration of test solution (mg / mL).

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