Method for determining six genotoxic impurities in rerugolil by high performance liquid chromatography-mass spectrometry

Abstract

The invention belongs to the technical field of drug detection, and particularly relates to a method for determining six genotoxic impurities in Rurugolil by high performance liquid chromatography-mass spectrometry. The method for determining the six genotoxic impurities in the Rurugolix by high performance liquid chromatography-mass spectrometry comprises the following steps: carrying out gradient elution by adopting a high performance liquid chromatography-mass spectrometry technology and taking octadecylsilane chemically bonded silica as a filler, a formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B as a chromatographic column, and carrying out gradient elution to obtain the six genotoxic impurities in the Rurugolix. A single-stage quadrupole mass spectrometry detector is adopted, and detection is carried out in an electrospray ionization (ESI) positive ion mode. The method for determining the six genotoxic impurities in the Rurugolil through high performance liquid chromatography-mass spectrometry is high in specificity and extremely high in sensitivity, and the six genotoxic impurities possibly existing in Rurugolil production can be effectively separated and accurately quantified, so that the product quality is better controlled.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection, and particularly relates to a method for the determination of 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry (HPLC-MS). Background Art

[0002] Relugolix is a gonadotropin-releasing hormone (GnRH) receptor antagonist, which is used for the bleeding and pain caused by uterine fibroids. In addition to treating uterine fibroids, this drug is also being studied for the pain caused by endometriosis and prostate cancer, etc. The chemical name of relugolix is 1-(4-{1-[(2,6-difluorophenyl)]-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea, with a molecular weight of 623.63, and the specific structural formula is shown as follows: 。

[0003] At present, the detection methods for genotoxic impurities in relugolix are relatively limited. For example, Patent CN116223685A discloses a method for the detection of key intermediates and related impurities of relugolix, which adopts high performance liquid chromatography. The chromatographic column used is YMC-Pack Pro C18, 150 mm×4.6 mm, 3 μm; the flow rate is 0.8 mL / min, the column temperature is 30 °C, the detection wavelength is 254 nm, the injection volume is 10 μL, mobile phase A is 20 mmol / L ammonium dihydrogen phosphate buffer solution (pH 7.0)-acetonitrile (70:30), and mobile phase B is acetonitrile for gradient elution. This detection method mainly studies the key intermediates of relugolix and 6 related impurities, but does not study the development of methods for genotoxic impurities that may be generated by the relevant processes in the finished product.

[0004] For example, CN118837477A, CN117969686A, and CN118980782A all disclose the detection methods for related substances in relugolix, all of which adopt high performance liquid chromatography. The determined impurities are all controlled as general impurities, the types of impurities are not comprehensive, and this method is controlled according to genotoxic impurities (the limit shall not exceed 27.5 ppm), and the sensitivity cannot meet the detection requirements.

[0005] The ultra-high performance liquid chromatography method for the determination of relugolix related substances is disclosed in CN118330071A. The ultra-high performance liquid chromatography conditions used in the determination include: the chromatographic column is filled with octadecylsilyl-bonded silica gel, perchloric acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is carried out. Twelve impurities generated in the production of relugolix are effectively separated and detected, thus effectively controlling the product quality, and impurities such as NOND, NOAC, and NOCD are disclosed. The impurities in this method are controlled as general impurities (not exceeding 0.1%), and this method is controlled as genotoxic impurities (the limit shall not exceed 27.5 ppm), and the sensitivity fails to meet the detection requirements. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects existing in the prior art, and provide a method for the determination of six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry, which has strong specificity and extremely high sensitivity, can effectively separate and accurately quantify six genotoxic impurities that may exist in the production of relugolix, and thus better controls the product quality.

[0007] The method for the determination of six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to the present invention, the six impurities are DQT, NOND, NOAC, NOCD, NOEE, and CNO, and the method includes: using high performance liquid chromatography-mass spectrometry technology, the chromatographic column is filled with octadecylsilyl-bonded silica gel, formic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, gradient elution is carried out, and a single quadrupole mass detector is used for detection in the positive ion mode of electrospray ionization (ESI).

[0008] The method for the determination of six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to the present invention, the method includes the following steps: (1) Preparation of impurity reference solution: Precisely weigh impurities DQT, NOND, NOAC, NOCD, NOEE, and CNO respectively, dissolve and dilute them with acetonitrile to prepare stock solutions of each impurity reference; precisely measure each stock solution of impurity reference, and dilute it with acetonitrile-formic acid aqueous solution to prepare each impurity reference solution; (2) Preparation of test solution: Precisely weigh relugolix, dissolve and dilute it with acetonitrile-formic acid aqueous solution, shake well, and use it as the test solution; (3) Detection: Inject the prepared impurity reference solutions and test solution into the high performance liquid chromatography-mass spectrometer for detection, and record the chromatogram; qualitatively analyze according to the retention time and mass number of each impurity in the impurity reference solutions and test solution, and calculate the content of each impurity by the external standard method.

[0009] The conditions for high performance liquid chromatography-mass spectrometry (HPLC-MS) also include: injection volume: 10 μL - 20 μL; the detector is a single quadrupole mass detector, column temperature: 33 °C - 37 °C; flow rate: 0.95 mL / min - 1.05 mL / min, more preferably 1.0 mL / min; in the positive ion mode of electrospray ionization (ESI), the mass-to-charge ratios of DQT, NOAC, NOND, CNO, NOEE, and NOCD are 112.2, 534.2, 641.2, 550.2, 562.2, and 581.2.

[0010] The chromatographic column is Waters Xbridge RP18, 4.6 mm × 250 mm, 5 µm. The volume ratio of mobile phase A to mobile phase B is (volume ratio 80:20) - (volume ratio 20:80).

[0011] The mass concentration of formic acid in the formic acid aqueous solution of mobile phase A is 0.08% - 0.12%, more preferably a 0.1% formic acid solution.

[0012] The conditions for gradient elution are: The first gradient elution time is 0 min, the proportion of mobile phase A is 80% - 70%, and the proportion of mobile phase B is 20% - 30%; The second gradient elution time is 25 min, the proportion of mobile phase A is 35% - 25%, and the proportion of mobile phase B is 65% - 75%; The third gradient elution time is 30 min, the proportion of mobile phase A is 35% - 25%, and the proportion of mobile phase B is 65% - 75%; The fourth gradient elution time is 31 min, the proportion of mobile phase A is 80% - 70%, and the proportion of mobile phase B is 20% - 30%; The fifth gradient elution time is 40 min, the proportion of mobile phase A is 80% - 70%, and the proportion of mobile phase B is 20% - 30%.

[0013] The volume ratio of acetonitrile - formic acid aqueous solution is 20:80 - 75:25, and the concentration of formic acid in the formic acid aqueous solution is 0.08% - 0.12%.

[0014] The concentration of each reference substance in each reference substance solution is 40 ng / mL - 60 ng / mL; The concentration of relugolix in the test solution is 1.5 mg / mL - 2.5 mg / mL.

[0015] The impurity DQT is 6-amino-3-hydroxypyridazine, NOND is propyl (2,6-difluorobenzyl)[4-(dimethylaminomethyl)-3-(6-methoxypyridazin-3-ylcarbamoyl)-5-(4-nitrophenyl)thiophen-2-yl]carbamate, NOAC is 2-[(2,6-difluorobenzyl)(ethylpropoxycarbonyl)amino]-4-(dimethylaminomethyl)-5-(4-nitrophenyl)thiophene-3-carboxylic acid, NOCD is 1-(2,6-difluorobenzyl)-5-(dimethylaminomethyl)-3-(6-methoxypyridazin-3-yl)-6-(4-nitrophenyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione, NOEE is ethyl 2-[(2,6-difluorobenzyl)n-propoxycarbonylamino]-4-((dimethylamino)methyl)-5-(4-nitrophenyl)thiophene-3-carboxylate, and CNO is 2-[(2,6-difluorobenzyl)(ethylpropoxycarbonyl)amino]-4-dimethylaminooxymethyl-5-(4-nitrophenyl)thiophene-3-carboxylic acid.

[0016] The structural formulas and chemical formulas of the respective genotoxic impurities are shown in Table 1.

[0017] Table 1 Genotoxic Impurities

[0018] The preferred elution conditions are shown in Table 2.

[0019] Table 2 Elution Conditions

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry (HPLC-MS) of the present invention has extremely high sensitivity, enabling accurate detection of genotoxic impurities such as DQT, NOND, NOAC, NOCD, NOEE and CNO, thereby effectively controlling the product quality.

[0021] (2) The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry (HPLC-MS) of the present invention has good resolution between impurities, good specificity, high sensitivity, good linear relationship, good repeatability, high precision, good durability, and more accurate and true results. Description of the Drawings

[0022] Figure 1 It is the liquid chromatography-mass spectrometry (LC-MS) chromatogram of the blank solution for the specificity test in Example 3 in the positive ion mode signal channel 1 (MSD1) and the positive ion mode signal channel 2 (MSD2).

[0023] Figure 2LC-MS chromatograms of the reference solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0024] Figure 3 LC-MS chromatograms of the test solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0025] Figure 4 LC-MS chromatograms of the spiked test solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0026] Figure 5 LC-MS chromatograms of the DQT positioning solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0027] Figure 6 LC-MS chromatograms of the NOND positioning solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0028] Figure 7 LC-MS chromatograms of the NOAC positioning solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0029] Figure 8 LC-MS chromatograms of the NOCD positioning solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0030] Figure 9 LC-MS chromatograms of the NOEE positioning solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0031] Figure 10 LC-MS chromatograms of the CNO positioning solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0032] Figure 11 LC-MS chromatograms of the detection limit solution of Example 4 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0033] Figure 12LC-MS chromatograms of the quantitation limit solution of Example 4 in the positive ion mode signal channel 1 (MSD1) and the positive ion mode signal channel 2 (MSD2). Detailed implementation manners

[0034] The present invention will be further described below in conjunction with specific embodiments.

[0035] The relugolix raw materials used in the examples are sourced from: Ruiyang Pharmaceutical Co., Ltd., batch number: 24013101. Other reagents, unless otherwise specified, are commercially available conventional raw materials.

[0036] Example 1 The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry comprises the following steps: (1) Preparation of impurity reference solution: DQT stock solution: Take impurity DQT, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 50 μg per 1 mL.

[0037] NOND stock solution: Take NOND, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 50 μg per 1 mL.

[0038] NOAC stock solution: Take NOAC, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 50 μg per 1 mL.

[0039] NOCD stock solution: Take NOCD, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 50 μg per 1 mL.

[0040] NOEE stock solution: Take NOEE, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 50 μg per 1 mL.

[0041] CNO stock solution: Take impurity CNO, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 50 μg per 1 mL.

[0042] DQT reference solution: Take the DQT stock solution and dilute it with acetonitrile - 0.1% formic acid solution (volume ratio 25:75) to a solution containing 50 ng per 1 mL.

[0043] NOND reference solution: Take the NOND stock solution and dilute it with acetonitrile - 0.1% formic acid solution (volume ratio 25:75) to a solution containing 50 ng per 1 mL.

[0044] NOAC reference solution: Take the NOAC stock solution and dilute it with acetonitrile - 0.1% formic acid solution (volume ratio 25:75) to a solution containing 50 ng per 1 mL.

[0045] NOCD reference solution: Take the NOCD stock solution and dilute it with acetonitrile - 0.1% formic acid solution (volume ratio 25:75) to a solution containing 50 ng per 1 mL.

[0046] NOEE reference solution: Take the NOEE stock solution and dilute it with acetonitrile - 0.1% formic acid solution (volume ratio 25:75) to a solution containing 50 ng per 1 mL.

[0047] CNO reference solution: Take the CNO stock solution and dilute it with acetonitrile - 0.1% formic acid solution (volume ratio 25:75) to a solution containing 50 ng per 1 mL.

[0048] (2) Preparation of test solution: Take 20 mg of relugolix, accurately weigh it, place it in a 10 mL volumetric flask, add acetonitrile - 0.1% formic acid solution (25:75) to dissolve and dilute to the mark, and shake well.

[0049] (3) Detection: Inject the prepared impurity reference solutions and test solution into a high performance liquid chromatography - mass spectrometry (HPLC - MS) instrument for detection, record the chromatogram, identify the impurities by the retention time of each impurity in the reference solution and test solution, and calculate the content of each impurity by the external standard method. The HPLC - MS conditions used include: injection volume of 10 μL - 20 μL; detector is a single - stage quadrupole mass detector; column temperature is 33 - 37 °C; flow rate is 0.95 - 1.05 mL / min; the chromatographic column is packed with octadecylsilyl bonded silica gel; mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile; gradient elution is carried out.

[0050] Method validation of the HPLC - MS determination method for the detection of genotoxic impurities in relugolix described in the present invention.

[0051] Example 2 (1) Diluent: Acetonitrile, acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75); Blank solution: Acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75); Test solution: Take about 20 mg of relugolix, accurately weigh it, place it in a 10 mL volumetric flask, add acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to dissolve and dilute to the mark, and shake well.

[0052] Reference solution: Take appropriate amounts of impurities DQT, NOND, NOAC, NOCD, NOEE and CNO, accurately weigh them, dissolve and dilute with acetonitrile to prepare a mixed stock solution with a concentration of 50 μg / mL for each impurity; take an appropriate amount of the mixed stock solution and dilute it with acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare a mixed solution with a concentration of 50 ng / mL for each impurity.

[0053] DQT Locating Solution: Take an appropriate amount of impurity DQT, dissolve it in acetonitrile and dilute to prepare a DQT stock solution with a concentration of 50 μg / mL; accurately measure an appropriate amount of the DQT stock solution and dilute it with an acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity locating solution with a concentration of 50 ng / mL.

[0054] NOND Locating Solution: Take an appropriate amount of NOND, dissolve it in acetonitrile and dilute to prepare a NOND stock solution with a concentration of 50 μg / mL; accurately measure an appropriate amount of the NOND stock solution and dilute it with an acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity locating solution with a concentration of 50 ng / mL.

[0055] NOAC Locating Solution: Take an appropriate amount of NOAC, dissolve it in acetonitrile and dilute to prepare a NOAC stock solution with a concentration of 50 μg / mL; accurately measure an appropriate amount of the NOAC stock solution and dilute it with an acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity locating solution with a concentration of 50 ng / mL.

[0056] NOCD Locating Solution: Take an appropriate amount of NOCD, dissolve it in acetonitrile and dilute to prepare a NOCD stock solution with a concentration of 50 μg / mL; accurately measure an appropriate amount of the NOCD stock solution and dilute it with an acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity locating solution with a concentration of 50 ng / mL.

[0057] NOEE Locating Solution: Take an appropriate amount of NOEE, dissolve it in acetonitrile and dilute to prepare a NOEE stock solution with a concentration of 50 μg / mL; accurately measure an appropriate amount of the NOEE stock solution and dilute it with an acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity locating solution with a concentration of 50 ng / mL.

[0058] CNO Locating Solution: Take an appropriate amount of impurity CNO, dissolve it in acetonitrile and dilute to prepare a CNO stock solution with a concentration of 50 μg / mL; accurately measure an appropriate amount of the CNO stock solution and dilute it with an acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity locating solution with a concentration of 50 ng / mL.

[0059] Reference Intermediate Solution: Accurately measure 1 mL each of the DQT stock solution, NOND stock solution, NOAC stock solution, NOCD stock solution, NOEE stock solution and CNO stock solution, place them in the same 100 mL volumetric flask, dilute to the mark with an acetonitrile - 0.1 wt% formic acid solution (25:75), and shake well.

[0060] Spiked test sample solution: Weigh accurately 20 mg of relugolix, place it in a 10 mL volumetric flask, add an appropriate amount of acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to dissolve it, accurately add 1 mL of the reference intermediate solution, and dilute to the mark with acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75), then shake well.

[0061] (2) Chromatographic and mass spectrometric conditions Chromatographic column: Waters Xbridge RP18, 4.6 mm × 250 mm, 5 µm; Column temperature: 35 °C; Mobile phase flow rate: 1.0 mL / min; Injection volume: 10 μL; Mobile phase: Use 0.1 wt% formic acid solution as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution as shown in Table 2.

[0062] Use a single - stage quadrupole mass spectrometer detector, and perform selected ion monitoring (SIM) in the positive ion mode of electrospray ionization (ESI). The information collected in the dual - channel is as follows: Positive ion mode signal channel 1: Mass - to - charge ratio (m / z) is 112.2 (impurity DQT), 534.2 (NOAC), 641.2 (NOND).

[0063] Positive ion mode signal channel 2: Mass - to - charge ratio (m / z) is 550.2 (impurity CNO), 562.2 (NOEE), 581.2 (NOCD).

[0064] Detection: Inject the prepared solution into a high - performance liquid chromatography - mass spectrometry (HPLC - MS) instrument for detection, and record the chromatogram.

[0065] (3) Calculation method: Content of each impurity (ppm) = weighed amount of reference substance × content of reference substance / dilution factor of reference substance × peak area of impurity / peak area of reference substance × dilution factor of test sample / weighed amount of test sample × 100%.

[0066] Example 3 (I) Specificity test: For the method in Example 2, inject blank solution, impurity location solutions, reference solution, test sample solution, and spiked test sample solution containing impurities as specificity solutions to investigate the specificity of the method. Through the specificity test, determine the retention time and resolution of the potential impurities under the determination conditions, and ensure that the impurities of interest can be effectively separated. The results of the specificity test are shown in Table 3, as Figures 1 to 10 shown: The blank solution is as Figure 1 shown, the reference solution is as Figure 2 shown, Figure 2In MSD1 (Channel 1), chromatographic peaks 1, 2, and 3 are DQT, NOND, and NOAC in sequence; in MSD2 (Channel 2), chromatographic peaks 4, 5, and 6 are NOCD, NOEE, and CNO in sequence.

[0067] The test solution is as Figure 3 shown, and the spiked test solution is as Figure 4 shown. The DQT calibration solution is as Figure 5 shown. The NOND calibration solution is as Figure 6 shown. The NOAC calibration solution is as Figure 7 shown. The NOCD calibration solution is as Figure 8 shown. The NOEE calibration solution is as Figure 9 shown. The CNO calibration solution is as Figure 10 shown. From the above detections, it can be seen that the main component in the spiked test solution can be effectively separated from adjacent impurities and between various impurities, and the specificity is good.

[0068] Table 3 Results of Specificity Test

[0069] Conclusion: The ratios of the retention times of each target impurity peak in the spiked test solution to the corresponding target peak retention time in the reference solution are all within the range of 0.98 - 1.02; the resolution between each target impurity peak in the spiked test solution is greater than 1.5; the specificity of this method is good.

[0070] Example 4 (2) Detection Limit and Quantitation Limit Tests: Take each impurity reference substance and adopt the stepwise dilution method. The concentration when the signal-to-noise ratio S / N ≥ 3 is used as the detection limit concentration; the concentration when the signal-to-noise S / N ≥ 10 is used as the quantitation limit concentration. The quantitation limit solution is injected continuously for 6 needles, and the RSD of the retention time should be ≤ 1.0%, and the RSD of the peak area should be ≤ 15%. The results of the quantitation limit and detection limit tests are shown in Tables 4 - 5. The LC-MS detection limit solution is as Figure 11 shown, and the quantitation limit solution is as Figure 12 shown.

[0071] Table 4 Results of Quantitation Limit Test

[0072] Table 5 Results of Detection Limit Test

[0073] Conclusion: When the quantitation limit solution is injected repeatedly for 6 times, the RSD of the retention time is less than 1.0%, the RSD of the peak area is less than 15%, and s / n is greater than 10; the s / n of the detection limit solution is greater than 3; the quantitation limit and detection limit are good.

[0074] Example 5 (III) Linearity and Range Test: The linearity of the method was achieved through the linearity of 6 solutions with different impurity concentrations. The peak area was plotted against the concentration, showing good linearity (linear correlation coefficient ≥ 0.995), and the determination range was determined simultaneously. The results of the linearity test for impurity DQT are shown in Table 6.

[0075] Table 6 Results of the Linearity Test for Impurity DQT

[0076] The results of the linearity test for NOND are shown in Table 7.

[0077] Table 7 Results of the Linearity Test for NOND

[0078] The results of the linearity test for NOAC are shown in Table 8.

[0079] Table 8 Results of the Linearity Test for NOAC

[0080] The results of the linearity test for NOCD are shown in Table 9.

[0081] Table 9 Results of the Linearity Test for NOCD

[0082] The results of the linearity test for NOEE are shown in Table 10.

[0083] Table 10 Results of the Linearity Test for NOEE

[0084] The results of the linearity test for impurity CNO are shown in Table 11.

[0085] Table 11 Results of the Linearity Test for Impurity CNO

[0086] Conclusion: The correlation coefficients of the linearity and range of 6 genotoxic impurities of relugolix are all greater than 0.990, showing good linearity. The ratio of the Y-axis intercept to the 100% response value is less than 30%. The external standard method with reference substance can be used for determination.

[0087] Example 6 (IV) Repeatability Test: Six spiked test solution samples were prepared in parallel, and the RSD of the recovery rates of each impurity in the spiked test solution was calculated to investigate the repeatability of the method. The results of each impurity in the repeatability test are shown in Table 12.

[0088] Table 12 Results of the Repeatability Test

[0089] Conclusion: The RSD of the impurity recoveries in 6 spiked test samples is ≤ 10%, meeting the requirements, indicating good repeatability of the method.

[0090] Example 7 (V) Precision Six spiked test samples were prepared in parallel under different dates, by different testers, and under different instrument conditions, and the RSD of the recoveries of each impurity in the spiked test samples was calculated to investigate the precision of the method. The results of each impurity in the precision test are shown in Table 13.

[0091] Table 13 Results of Precision Test

[0092] Conclusion: For the 12 spiked test samples, the RSDs of the recoveries of each impurity measured are all less than 15%, indicating good precision of the method.

[0093] Example 8 (VI) Accuracy The accuracy of the method was achieved through 3 solutions with different impurity concentrations, and the RSD of the recoveries of each impurity in the spiked test samples was calculated. The results of the accuracy test are shown in Tables 14 - 19.

[0094] Table 14 Results of Recovery Test for Impurity DQT

[0095] Table 15 Results of Recovery Test for NOND

[0096] Table 16 Results of Recovery Test for NOAC

[0097] Table 17 Results of Recovery Test for NOCD

[0098] Table 18 Results of Recovery Test for NOEE

[0099] Table 19 Results of Recovery Test for Impurity CNO

[0100] Conclusion: It can be seen from the test results that for impurities DQT, NOND, NOAC, NOCD, NOEE, and impurity CNO within the concentration range of 50% - 150% of the limit concentration, both the single - value and average recovery rates are between 70% - 130%, the RSD is less than 15%, and the method has good accuracy.

[0101] Example 9 (IX) Robustness test: Based on Example 1, with other conditions remaining unchanged, by slightly adjusting the chromatographic conditions, the determination results of genotoxic impurities under different conditions are consistent, indicating good robustness. The results measured under different chromatographic conditions are shown in Table 20.

[0102] Table 20 Results of the robustness test

[0103] Conclusion: By slightly changing the column temperature (standard condition ±2°C), flow rate (standard condition ±0.05 mL / min), and under different chromatographic column conditions, the RSD of the impurity content is less than 15%, meeting the requirements, and the method has good robustness.

[0104] From the above verification results, it can be known that the determination method of the present invention is high - performance liquid chromatography - mass spectrometry (HPLC - MS), which can accurately determine 6 genotoxic impurities DQT, NOND, NOAC, NOCD, NOEE, and CNO in relugolix, and the quantification limit is much lower than the limit (27.5 ppm), thus effectively controlling the product quality; this method has high sensitivity, strong specificity, good precision (instrument precision, repeatability, intermediate precision), linearity, accuracy, solution stability, and robustness, and the results are stable and reliable. Therefore, it can be used for the quality control of relugolix, providing effective guarantee for the quality of the final product, and filling the blank of detecting genotoxic impurities in relugolix by using high - performance liquid chromatography - mass spectrometry.

Claims

1. A method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry, characterized in that: The six impurities are DQT, NOND, NOAC, NOCD, NOEE, and CNO. The method comprises: using high performance liquid chromatography-mass spectrometry technology, using octadecylsilane bonded silica gel as a filler for the chromatographic column, using formic acid aqueous solution as mobile phase A, using acetonitrile as mobile phase B, gradient elution, using a single-stage quadrupole mass spectrometer detector, and detecting in a positive ion mode of electrospray ionization.

2. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The method comprises the following steps: (1) Preparation of impurity reference solution: Accurately weigh impurities DQT, NOND, NOAC, NOCD, NOEE and CNO respectively, dissolve them in acetonitrile and dilute them to prepare the stock solutions of each impurity reference substance; accurately measure the stock solutions of each impurity reference substance, dilute them with acetonitrile-formic acid aqueous solution to prepare the solutions of each impurity reference substance; (2) Preparation of test solution: Accurately weigh relugolix, dissolve and dilute with acetonitrile-formic acid aqueous solution, and shake well to prepare the test solution; (3) Detection: Inject the prepared impurity reference solution and test solution into a high performance liquid chromatography-mass spectrometer for detection and record the chromatogram. The impurities in the impurity reference solution and test solution are qualitatively identified by their retention time and mass number, and the content of each impurity is calculated by the external standard method.

3. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The conditions for using high performance liquid chromatography-mass spectrometry also include: injection volume: 10μL~20μL; the detector is a single-stage quadrupole mass spectrometer detector, column temperature: 33℃~37℃; flow rate: 0.95mL / min~1.05mL / min; in the positive ion mode of electrospray ionization, the mass-to-charge ratios of DQT, NOAC, NOND, CNO, NOEE, and NOCD are selected as 112.2, 534.2, 641.2, 550.2, 562.2, and 581.

2.

4. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 3, characterized in that: The chromatographic column was Waters Xbridge RP18, 4.6 mm × 250 mm, 5 µm.

5. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mass concentration of formic acid in the formic acid aqueous solution is 0.08% to 0.12%.

6. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The conditions for gradient elution were: The first gradient elution time is 0 min, mobile phase A accounts for 80% to 70%, and mobile phase B accounts for 20% to 30%; The second gradient elution time is 25 min, with mobile phase A accounting for 35% to 25% and mobile phase B accounting for 65% to 75%; The third gradient elution time is 30 min, mobile phase A accounts for 35% to 25%, and mobile phase B accounts for 65% to 75%; The fourth gradient elution time was 31 min, with mobile phase A accounting for 80% to 70% and mobile phase B accounting for 20% to 30%; The fifth gradient elution time is 40 min, with mobile phase A accounting for 80% to 70% and mobile phase B accounting for 20% to 30%.

7. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 2, characterized in that: The volume ratio of acetonitrile-formic acid aqueous solution is 20:80-75:25, and the concentration of formic acid in the formic acid aqueous solution is 0.08%-0.12%.

8. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 2, characterized in that: The concentration of each reference substance in each impurity reference substance solution is 40 ng / mL to 60 ng / mL.

9. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 2, characterized in that: The concentration of relugolix in the test solution is 1.5 mg / mL to 2.5 mg / mL.

10. The method for determining six genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The impurity DQT is 6-amino-3-hydroxypyridazine, NOND is (2,6-difluorobenzyl) [4-dimethylaminomethyl-3- (6-methoxypyridazine-3-ylcarbamoyl) -5- (4-nitrophenyl) thiophene-2-yl] propyl carbamate, NOAC is 2- [(2,6-difluorobenzyl) (ethylpropoxycarbonyl) amino] -4-dimethylaminomethyl-5- (4-nitrophenyl) thiophene-3-carboxylic acid, NOCD is 1- (2,6-difluorobenzyl) -5-dimethylaminomethyl The invention relates to 2-[(2,6-difluorobenzyl)-n-propoxycarbonylamino]-4-((dimethylamino)methyl)-5-(4-nitrophenyl)thiophene-3-carboxylic acid ethyl ester and 2-[(2,6-difluorobenzyl)(ethylpropoxycarbonyl)amino]-4-dimethylaminooxymethyl-5-(4-nitrophenyl)thiophene-3-carboxylic acid ethyl ester.

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