Method for Detecting Genotoxic Impurities in Rilpivirine Hydrochloride

Abstract

The present invention belongs to the technical field of pharmaceutical detection, and particularly relates to a method for detecting genotoxic impurities in ledipasvir hydrochloride. The method for detecting genotoxic impurities in ledipasvir hydrochloride uses high performance liquid chromatography for detection. Using 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline as reference substances, methanol as the diluent, and the external standard method to calculate the contents of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline in the sample. The detection wavelength is 200-220 nm. The chromatographic column uses octadecylsilane chemically bonded silica as the filler. Mobile phase A is ammonia water, mobile phase B is acetonitrile, and gradient elution is performed. The present invention fills the blank of analyzing these two genotoxic impurities, 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline, by high performance liquid chromatography. Compared with the traditional liquid chromatography-mass spectrometry detection method, this method is simple to operate, convenient and efficient, has a wide application range, higher sensitivity, and ensures the safety of drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical detection, and particularly relates to a method for detecting genotoxic impurities in rilpivirine hydrochloride. Background Art

[0002] Genotoxic impurities are a class of impurities that can react with DNA, cause DNA damage, induce gene mutations at very low levels, and may be carcinogenic. Due to their strong toxicity, which poses a strong threat to the safety of drug use, regulatory agencies in various countries such as ICH, FDA, and EMA have had more explicit requirements for genotoxic impurities in recent years. More and more pharmaceutical companies have focused on the control and detection of genotoxic impurities during the new drug R & D process.

[0003] 2,6-Dimethylaniline and 2,6-dimethyl-4-bromoaniline are respectively the starting material and intermediate of the starting materials in the synthesis of rilpivirine hydrochloride, and are genotoxic impurities with warning structures.

[0004] The structural formula of 2,6-dimethylaniline is as follows:

[0005]

[0006] The structural formula of 2,6-dimethyl-4-bromoaniline is as follows:

[0007]

[0008] Referring to the ICH guideline M7, both 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline are classified as category 3 according to mutagenicity and carcinogenicity, with a limit ≤ 1.5 μg / Day. There is no relevant report on the detection method for this genotoxic impurity. To ensure the drug safety of rilpivirine hydrochloride, a method is needed to separate and detect its genotoxic impurities. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a method for detecting genotoxic impurities in rilpivirine hydrochloride, filling the blank of analyzing these two genotoxic impurities, 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline, by high performance liquid chromatography method. Compared with the traditional liquid chromatography-mass spectrometry detection method, this method is simple, convenient, highly efficient, has a wide application range, and higher sensitivity. The sensitivity of 2,6-dimethylaniline can reach 32 ppm, and the sensitivity of 2,6-dimethyl-4-bromoaniline can reach 30 ppm, ensuring the safety of the drug.

[0010] The method for detecting genotoxic impurities in ledipasvir hydrochloride according to the present invention uses high performance liquid chromatography for detection. Using 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline as reference substances, methanol as the diluent, and calculating the contents of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline in the sample by the external standard method. The detection wavelength is 200 - 220 nm. The chromatographic column uses octadecylsilane bonded silica as the packing material. Mobile phase A is ammonia water, and mobile phase B is acetonitrile. The elution program is gradient elution;

[0011] The gradient elution conditions are as follows:

[0012] For the first gradient elution, the time is 0 min, the proportion of mobile phase A is 55 - 65%, and the proportion of mobile phase B is 35 - 45%;

[0013] For the second gradient elution, the time is 12 min, the proportion of mobile phase A is 55 - 65%, and the proportion of mobile phase B is 35 - 45%;

[0014] For the third gradient elution, the time is 15 min, the proportion of mobile phase A is 45 - 55%, and the proportion of mobile phase B is 45 - 55%;

[0015] For the fourth gradient elution, the time is 25 min, the proportion of mobile phase A is 45 - 55%, and the proportion of mobile phase B is 45 - 55%;

[0016] For the fifth gradient elution, the time is 40 min, the proportion of mobile phase A is 0 - 10%, and the proportion of mobile phase B is 90 - 100%;

[0017] For the sixth gradient elution, the time is 41 min, the proportion of mobile phase A is 55 - 65%, and the proportion of mobile phase B is 35 - 45%;

[0018] For the seventh gradient elution, the time is 55 min, the proportion of mobile phase A is 55 - 65%, and the proportion of mobile phase B is 35 - 45%.

[0019] Preferably, the concentration of ammonia water in mobile phase A is 0.05 - 1.0 wt%, and the pH is adjusted to 5.0 - 7.5 with phosphoric acid.

[0020] Preferably, the chromatographic column size is 4.6×250 mm, and the average particle size of the packing material is 5 μm.

[0021] Preferably, the column temperature is 30 - 35°C.

[0022] Preferably, the flow rate of the mobile phase is 0.8 - 1.2 mL / min.

[0023] Preferably, the injection volume is 10 - 20 μl.

[0024] In a most preferred embodiment, the chromatographic conditions are as follows:

[0025] Chromatographic column: Size 4.6×250 mm, using octadecylsilyl silica gel as the packing material, and the average particle size of the packing material is 5 μm;

[0026] Column temperature: 30 °C;

[0027] Mobile phase flow rate: 1.0 mL / min;

[0028] Sample injection volume: 20 μl;

[0029] Detection wavelength: 310 nm;

[0030] Mobile phase: Mobile phase A is ammonia water with a concentration of 0.1 wt%, and the pH is adjusted to 6.5 with phosphoric acid; Mobile phase B is acetonitrile; Gradient elution;

[0031] Gradient elution conditions:

[0032] The first gradient elution time is 0 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%;

[0033] The second gradient elution time is 12 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%;

[0034] The third gradient elution time is 15 min, the proportion of mobile phase A is 50%, and the proportion of mobile phase B is 50%;

[0035] The fourth gradient elution time is 25 min, the proportion of mobile phase A is 50%, and the proportion of mobile phase B is 50%;

[0036] The fifth gradient elution time is 40 min, the proportion of mobile phase A is 0%, and the proportion of mobile phase B is 100%;

[0037] The sixth gradient elution time is 41 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%;

[0038] The seventh gradient elution time is 55 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%.

[0039] Preferably, the detection method includes the following steps:

[0040] (1) Prepare a blank solvent:

[0041] Use the diluent as the blank solvent;

[0042] (2) Prepare a reference solution:

[0043] Using 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline as reference substances, and methanol in the diluent as the solvent, prepare a reference solution containing 55 ng of 2,6-dimethylaniline and 55 ng of 2,6-dimethyl-4-bromoaniline per 1 mL;

[0044] (3) Prepare the test solution:

[0045] Take the test sample of rilpivirine hydrochloride, weigh accurately, dissolve and dilute with the diluent methanol to prepare a solution containing 1.0 mg of the test sample per 1 mL as the test solution;

[0046] (4) Detection:

[0047] Precisely measure the blank solvent, reference solution, and test solution respectively, inject them into the high-performance liquid chromatograph, record the chromatogram, and separate rilpivirine hydrochloride and genotoxic impurities under the same high-performance liquid chromatography conditions.

[0048] The genotoxic impurities are 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline. The structural formula of 2,6-dimethylaniline is as follows:

[0049]

[0050] The structural formula of 2,6-dimethyl-4-bromoaniline is as follows:

[0051]

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0053] The present invention uses high-performance liquid chromatography to detect the genotoxic impurities of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline in rilpivirine hydrochloride. The operation is simple, the detection speed is fast, the detection equipment is relatively common, the detection cost is low, the separation efficiency is high, the sensitivity is high, the specificity is strong, the accuracy is high, and the durability is good. By detecting the genotoxic impurities of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline in rilpivirine hydrochloride, the quality of the product can be better controlled. Description of the Drawings

[0054] Figure 1 It is the HPLC chromatogram of the blank solvent of the present invention;

[0055] Figure 2 It is the HPLC chromatogram of the reference solution of the present invention;

[0056] Figure 3 It is the HPLC chromatogram of the blank solvent in Example 1 of the present invention;

[0057] Figure 4 It is the HPLC chromatogram of the positioning solution in Example 1 of the present invention;

[0058] Figure 5 It is the HPLC chromatogram of the test solution in Example 1 of the present invention;

[0059] Figure 6 This is the HPLC chromatogram of the impurity test sample mixed solution in Example 1 of the present invention;

[0060] Figure 7 This is the superposition comparison diagram in Example 1 of the present invention;

[0061] Figure 8 This is the HPLC chromatogram of the quantitation limit solution in Example 2 of the present invention;

[0062] Figure 9 This is the HPLC chromatogram of the detection limit solution in Example 2 of the present invention;

[0063] Figure 10 This is the linear regression diagram of 2,6-dimethylaniline in Example 3 of the present invention;

[0064] Figure 11 This is the linear regression diagram of 2,6-dimethyl-4-bromoaniline in Example 3 of the present invention;

[0065] Figure 12 This is the HPLC chromatogram of the spiked test sample solution under the standard conditions in Example 8 of the present invention;

[0066] Figure 13 This is the HPLC chromatogram of the spiked test sample solution under the condition of a flow rate of 0.9 mL / min in Example 8 of the present invention;

[0067] Figure 14 This is the HPLC chromatogram of the spiked test sample solution under the condition of a flow rate of 1.2 mL / min in Example 8 of the present invention;

[0068] Figure 15 This is the HPLC chromatogram of the spiked test sample solution under the condition of Column 1 in Example 8 of the present invention;

[0069] Figure 16 This is the HPLC chromatogram of the spiked test sample solution under the condition of Column 2 in Example 8 of the present invention. Detailed implementation manners

[0070] The present invention will be further described below in conjunction with examples. The raw materials used in the examples are all commercially available conventional raw materials unless otherwise specified; the process methods used in the examples are all conventional methods in the art unless otherwise specified.

[0071] In the following examples, the high performance liquid chromatography conditions are as follows:

[0072] Chromatographic column: size 4.6×250 mm, using octadecylsilane bonded silica gel as the filler, and the average particle size of the filler particles is 5 μm;

[0073] Column temperature: 30°C;

[0074] Mobile phase flow rate: 1.0 mL / min;

[0075] Sample injection volume: 20 μl;

[0076] Detection wavelength: 310 nm;

[0077] Mobile phase: Mobile phase A is ammonia water with a concentration of 0.1 wt%, and the pH value is adjusted to 6.5 with phosphoric acid; Mobile phase B is acetonitrile; Gradient elution;

[0078] Gradient elution conditions:

[0079] The first gradient elution time is 0 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%;

[0080] The second gradient elution time is 12 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%;

[0081] The third gradient elution time is 15 min, the proportion of mobile phase A is 50%, and the proportion of mobile phase B is 50%;

[0082] The fourth gradient elution time is 25 min, the proportion of mobile phase A is 50%, and the proportion of mobile phase B is 50%;

[0083] The fifth gradient elution time is 40 min, the proportion of mobile phase A is 0%, and the proportion of mobile phase B is 100%;

[0084] The sixth gradient elution time is 41 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%;

[0085] The seventh gradient elution time is 55 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%;

[0086] Diluent: Methanol.

[0087] (1) Regarding the selection of detection wavelength:

[0088] Perform ultraviolet spectrum scanning on 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline: Take appropriate amounts of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline reference substances, dissolve and dilute them with methanol to make solutions of 20 μg / mL respectively, take each solution and scan it from 400 nm to 200 nm, and the results are shown in Table 1.

[0089] Table 1 Maximum absorption wavelengths of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline

[0090] Name Maximum absorption wavelength / Absorbance value 2,6-Dimethylaniline A208nm / 0.91; A241nm / 0.42; A293nm / 0.31 2,6-Dimethyl-4-bromoaniline A208nm / 0.98; A232nm / 0.31; A280nm / 0.21 Blank solvent /

[0091] As can be seen from Table 1, both 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline have relatively large ultraviolet absorptions at wavelengths near 208 nm, indicating that 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline can be detected by HPLC with an ultraviolet detector. Considering the end absorption, 210 nm was selected as the detection wavelength.

[0092] (2) Selection of the diluent (solvent):

[0093] The solubility of this compound, rilpivirine hydrochloride, is slightly soluble in methanol, the best among conventional solvents, and almost insoluble in ethyl acetate, acetonitrile, and water. Considering the low limit of genotoxic impurities and the need for a relatively high concentration of the test substance, methanol was selected as the solvent for preparing the test substance.

[0094] (3) Selection of mobile phase B (organic phase):

[0095] The commonly used organic phases in high-performance liquid chromatography are methanol, acetonitrile, and tetrahydrofuran. The detection wavelength of the compound is 210 nm. Since methanol and tetrahydrofuran have end absorption interference, acetonitrile was selected as the organic phase.

[0096] (4) Selection of mobile phase A and pH regulator:

[0097] The main structure of rilpivirine hydrochloride is weakly basic. For weakly basic compounds within the pH range of 2 - 9, the retention time follows the principle of increasing with the increase of pH.

[0098] Consider the following factors:

[0099] ① Solvent effect. The diluent was selected as pure organic phase methanol. Therefore, the initial proportion of the organic phase in the gradient elution program is relatively low, which may cause solvent effect for the peaks with earlier elution times, resulting in poor peak shapes and affecting quantitative determination.

[0100] ② Interference factors of the test substance. To avoid the interference of the rilpivirine hydrochloride peak on the quantitative determination of genotoxic impurities, the rilpivirine hydrochloride peak needs to elute after the genotoxic impurity peaks. Considering the above factors, the pH of the mobile phase was selected to be 5.0 - 7.5.

[0101] The main structure of rilpivirine hydrochloride is weakly basic. Basic compounds are prone to peak tailing due to the silanol effect. Therefore, an appropriate amount of basic tailing agent was selected. At low wavelengths, ammonia water introduces fewer impurities than other basic tailing agents. So ammonia water was selected as the basic tailing agent. After adding ammonia water, an acidic pH regulator was needed to adjust the pH to 5.0 - 7.5. Compared with other acidic regulators such as acetic acid, formic acid, and trifluoroacetic acid, phosphoric acid has the advantages of low cut-off wavelength and fewer introduced impurities. Therefore, phosphoric acid was selected as the acidic pH regulator.

[0102] (5) Selection of gradient elution program:

[0103] Consider the following factors:

[0104] ① The initial organic phase ratio is higher than 20%;

[0105] ② The elution peaks of the two genotoxic impurities should appear earlier than the main component rilpivirine hydrochloride;

[0106] ③ The gradient elution program should not have gradient peaks that interfere with the quantitative determination of genotoxic impurities;

[0107] ④ The baseline during the elution period of the two genotoxic impurities must be stable;

[0108] ⑤ The peak shapes of genotoxic impurities are reasonable, and the sensitivity must meet the requirements of quantitative determination.

[0109] Considering the above factors comprehensively, the gradient elution program is determined to be the above conditions.

[0110] Examples 1-7 respectively test the specificity, limit of quantitation and limit of detection, linearity and range, accuracy, repeatability, intermediate precision, solution stability and durability of this detection method. The preparation method of the test sample solution is as follows:

[0111] (1) Preparation of blank solvent:

[0112] Use diluted methanol as the blank solvent;

[0113] (2) Preparation of stock solution of reference substance:

[0114] Using 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline as reference substances and diluted methanol as the solvent, prepare a solution containing 550 ng of 2,6-dimethylaniline and 550 ng of 2,6-dimethyl-4-bromoaniline per 1 mL as the stock solution of reference substance;

[0115] (3) Preparation of reference substance solution:

[0116] Using 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline as reference substances and diluted methanol as the solvent, prepare a mixed reference substance solution containing 55 ng of 2,6-dimethylaniline and 55 ng of 2,6-dimethyl-4-bromoaniline per 1 mL;

[0117] (4) Preparation of test solution:

[0118] Take the test sample of rilpivirine hydrochloride, weigh it accurately, dissolve and dilute it with diluted methanol to prepare a solution containing 1.0 mg of the test sample per 1 mL as the test solution;

[0119] (5) Preparation of spiked test solution:

[0120] Accurately weigh 10 mg of the test sample of rilpivirine hydrochloride, place it in a 10 mL volumetric flask, accurately add 1 mL of the reference stock solution, add methanol as the diluent, ultrasonically dissolve and dilute to the mark, and shake well to obtain the spiked test sample solution.

[0121] Take 10 μL of the blank solvent and 10 μL of the reference solution respectively for high performance liquid chromatography detection, record the chromatogram. As Figure 1-2 shown, obtain the peak elution time and peak area of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline in the reference solution.

[0122] Example 1

[0123] Specificity:

[0124] Localization solution: Take appropriate amounts of the reference substances of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline, add methanol and ultrasonically dissolve and dilute to prepare a localization solution containing about 2.75 μg of each of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline per 1 mL.

[0125] Impurity test sample mixed solution: Take appropriate amounts of the test sample of rilpivirine hydrochloride, the reference substances of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline, add methanol and ultrasonically dissolve and dilute to prepare an impurity test sample mixed solution containing about 2.75 μg of each of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline and 1 mg of rilpivirine hydrochloride per 1 mL.

[0126] Take 10 μL each of the blank solvent, the localization solution, the test sample solution and the impurity test sample mixed solution and inject them into the high performance liquid chromatograph respectively, record the chromatogram. As Figure 3-6 shown, Figure 7 is the superimposed comparison chart of each chromatogram.

[0127] It can be seen from Figure 3-7 that the resolution of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline is good, and the matrix in the blank solvent and the test sample does not interfere with the determination of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline, indicating that the detection method of the present invention has good specificity.

[0128] Example 2

[0129] Quantitation limit and detection limit test:

[0130] Take appropriate amounts of the reference substances of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline, add methanol to dissolve and dilute to prepare the corresponding solutions. Take 10 μL and inject it into the high performance liquid chromatograph. Take the concentration of the solution when S / N≈10 as the quantitation limit concentration, dilute the quantitation limit solution by 2 - 3 times, and take the concentration of the solution when S / N≈3 as the detection limit concentration. At the same time, investigate the precision of the quantitation limit.

[0131] The chromatograms of the quantitation limit solution and the detection limit solution are as Figure 8-9 shown. The results show that the quantitation limit concentration of 2,6-dimethylaniline is 0.03138 μg / mL, and the detection limit concentration is 0.01569 μg / mL; the quantitation limit concentration of 2,6-dimethyl-4-bromoaniline is 0.02974 μg / mL, and the detection limit concentration is 0.01487 μg / mL.

[0132] Example 3

[0133] Linearity and range test:

[0134] Accurately measure an appropriate amount of the reference stock solution, and sequentially dilute it with methanol to prepare a series of reference solutions containing 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline at concentrations of 0.0275 μg / mL, 0.04112 μg / mL, 0.055 μg / mL, 0.0825 μg / mL, and 0.11 μg / mL. Take 10 μL of each of the above series of reference solutions and inject them into the high performance liquid chromatograph respectively. Record the chromatograms. Using the concentration as the abscissa and the peak areas of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline as the ordinates respectively, perform linear regression by the least squares method. The linear regression is as Figure 10-11 shown, and the results are shown in Table 2-3.

[0135] Table 2 Results of the linear relationship test of 2,6-dimethylaniline

[0136]

[0137] Table 3 Results of the linear relationship test of 2,6-dimethyl-4-bromoaniline

[0138]

[0139] Example 4

[0140] Accuracy test:

[0141] Preparation of the recovery solution:

[0142] Accurately weigh 10 mg of the test sample, place it in a 10 mL volumetric flask, add an appropriate amount of the reference stock solution according to the 50% level of the limit, dissolve it ultrasonically with methanol and dilute it to the scale, shake well, and prepare 3 parallels as the 50% recovery solution.

[0143] Accurately weigh 10 mg of the test sample, place it in a 10 mL volumetric flask, add an appropriate amount of the reference stock solution according to the 100% level of the limit, dissolve it ultrasonically with methanol and dilute it to the scale, shake well, and prepare 3 parallels as the 100% recovery solution.

[0144] Accurately weigh 10 mg of the test sample, place it in a 10 mL volumetric flask, add an appropriate amount of the reference stock solution at the level of 150% of the limit, dissolve it ultrasonically with methanol and dilute to the mark, shake well, and prepare 3 portions in parallel as the 150% recovery solution.

[0145] Take 10 μL each of the blank solvent, reference solution, test solution, and each recovery solution, inject them into the high performance liquid chromatograph respectively, and record the chromatograms. Calculate the total measured amounts of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline by the external standard method based on the peak areas, calculate the recoveries, and the test results are shown in Table 4 5.

[0146] Table 4 Test Results of the Recovery of 2,6-Dimethylaniline

[0147]

[0148] Table 5 Test Results of the Recovery of 2,6-Dimethyl-4-bromoaniline

[0149]

[0150]

[0151] Example 5

[0152] Repeatability test:

[0153] Prepare 6 portions of spiked test solutions in parallel. Take 10 μL each of the blank solvent, reference solution, and spiked test solution, inject them into the high performance liquid chromatograph respectively, and record the chromatograms. Calculate the contents of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline by the external standard method based on the peak areas, and calculate the RSD. The test results are shown in Table 6.

[0154] Table 6 Test Results of Repeatability

[0155]

[0156] Example 6

[0157] Intermediate precision test:

[0158] Change different times, different instruments, and different personnel, and prepare 6 portions of spiked test solutions in parallel.

[0159] Take 10 μL each of the blank solution, reference solution, and spiked test solution, inject them into the high performance liquid chromatograph respectively, and record the chromatograms. Calculate the contents of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline by the external standard method based on the peak areas, calculate the RSD (n = 12), and investigate the intermediate precision of the method. The test results are shown in Table 7.

[0160] Table 7 Test Results of Intermediate Precision

[0161]

[0162] Example 7

[0163] Solution stability test:

[0164] Take 10 μL of the spiked test sample solution after standing at room temperature for 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, inject it into a high performance liquid chromatograph, record the chromatogram, and calculate the RSD value of the change in the peak area of each genotoxic impurity. The test results are shown in Table 8.

[0165] Table 8 Results of solution stability test

[0166]

[0167]

[0168] Example 8

[0169] Robustness test:

[0170] In order to investigate the tolerance of the method of the present invention for the detection of this product when there are minor changes in the chromatographic conditions, the chromatographic condition robustness was investigated, and the specific parameter changes are shown in Table 9.

[0171] Table 9 Variation parameters of chromatographic conditions

[0172] Chromatographic parameters Standard conditions Range to be verified Flow rate 1.0 mL / min 0.9 mL / min~1.2 mL / min Mobile phase pH 6.5 6.3～6.7 Chromatographic column C18 Chromatographic columns of different brands

[0173] Take 10 μL of the blank solution, reference solution, and spiked test sample solution respectively, inject them into a high performance liquid chromatograph, and record the chromatogram. Calculate the contents of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline by the external standard method based on the peak area. The chromatogram of the spiked test sample solution is as Figure 12-16 shown, and the test results are shown in Table 10.

[0174] Table 10 Results of robustness test

[0175]

Claims

1. A method for detecting genotoxic impurities in ledipasvir hydrochloride, characterized in that: Determined by high performance liquid chromatography (HPLC). Using 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline as reference substances, methanol as the diluent, and the external standard method to calculate the contents of 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline in the sample; The chromatographic conditions are as follows: Chromatographic column: Size 4.6×250 mm, using octadecylsilyl bonded silica gel as the packing material, and the average particle size of the packing material particles is 5 μm; Column temperature: 30 °C; Mobile phase flow rate: 1.0 mL / min; Injection volume: 20 μL; Detection wavelength: 210 nm; Mobile phase: Mobile phase A is ammonia water with a concentration of 0.1 wt%, adjusted to pH 6.5 with phosphoric acid; Mobile phase B is acetonitrile; Gradient elution; Gradient elution conditions: The first gradient elution time is 0 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%; The second gradient elution time is 12 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%; The third gradient elution time is 15 min, the proportion of mobile phase A is 50%, and the proportion of mobile phase B is 50%; The fourth gradient elution time is 25 min, the proportion of mobile phase A is 50%, and the proportion of mobile phase B is 50%; The fifth gradient elution time is 40 min, the proportion of mobile phase A is 0%, and the proportion of mobile phase B is 100%; The sixth gradient elution time is 41 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%; The seventh gradient elution time is 55 min, the proportion of mobile phase A is 60%, and the proportion of mobile phase B is 40%.

2. The method for detecting genotoxic impurities in ledipasvir hydrochloride according to claim 1, characterized in that: The detection method includes the following steps: (1) Preparation of blank solvent: Using the diluent as the blank solvent; (2) Preparation of reference substance solution: Using 2,6-dimethylaniline and 2,6-dimethyl-4-bromoaniline as reference substances, methanol as the diluent, and preparing a reference substance solution containing 55 ng of 2,6-dimethylaniline and 55 ng of 2,6-dimethyl-4-bromoaniline per 1 mL; (3) Preparation of test solution: Accurately weigh the test substance of rilpivirine hydrochloride, dissolve and dilute it with the diluent methanol to prepare a solution containing 1.0 mg of the test substance per 1 mL as the test solution; (4) Detection: Precisely measure 10 μL of the blank solvent, reference substance solution, and test solution respectively, inject them into the high performance liquid chromatograph, record the chromatogram, and separate rilpivirine hydrochloride and genotoxic impurities under the same high performance liquid chromatographic conditions.

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