Method for the determination of 6 genotoxic impurities in relugolix by high performance liquid chromatography - mass spectrometry

Abstract

The present invention belongs to the technical field of drug detection, and specifically relates to a method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry (HPLC-MS). The method for determining 6 genotoxic impurities in relugolix by HPLC-MS according to the present invention includes: using HPLC-MS technology, with a chromatographic column filled with octadecylsilyl-bonded silica gel, formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution, and using a single quadrupole mass detector for detection in the positive ion mode of electrospray ionization (ESI). The method for determining 6 genotoxic impurities in relugolix by HPLC-MS provided by the present invention has strong specificity and extremely high sensitivity, can effectively separate and accurately quantify 6 genotoxic impurities that may exist in the production of relugolix, thereby better controlling the product quality.

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. Background Art

[0002] Relugolix is a gonadotropin-releasing hormone (GnRH) receptor antagonist, which is used for bleeding and pain caused by uterine fibroids. In addition to treating uterine fibroids, the drug is also being studied for 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] .

[0004] 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, 150mm×4.6mm, 3μm; the flow rate is 0.8mL / min, the column temperature is 30°C, the detection wavelength is 254nm, the injection volume is 10µ1, mobile phase A is 20mmol / L ammonium dihydrogen phosphate buffer solution (pH value 7.0)-acetonitrile (70:30), and mobile phase B is acetonitrile for gradient elution. This detection method mainly studies the key intermediates and 6 related impurities of relugolix, but does not study the development method for genotoxic impurities that may be generated by the relevant processes in the finished product.

[0005] 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 27.5ppm), and the sensitivity cannot meet the detection requirements.

[0006] The ultra-high performance liquid chromatography determination method of relugolix related substances is disclosed in CN118330071A. The ultra-high performance liquid chromatography conditions adopted in the determination include: the chromatographic column is filled with octadecylsilane-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 to effectively separate and detect 12 impurities generated in the production of relugolix, 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

[0007] 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 determining 6 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 6 genotoxic impurities that may exist in the production of relugolix, and thus better controls the product quality.

[0008] The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to the present invention, the 6 impurities are DQT, NOND, NOAC, NOCD, NOEE, and CNO, and the method includes: adopting high performance liquid chromatography-mass spectrometry technology, the chromatographic column is filled with octadecylsilane-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 adopted for detection in the positive ion mode of electrospray ionization (ESI).

[0009] The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to the present invention, the method includes the following steps:

[0010] (1) Preparation of impurity reference solution:

[0011] 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;

[0012] (2) Preparation of test solution:

[0013] Precisely weigh relugolix, dissolve and dilute it with acetonitrile-formic acid aqueous solution, shake well, and use it as the test solution;

[0014] (3) Detection: Inject the prepared impurity reference solutions and the test solution into a high performance liquid chromatography-mass spectrometry (HPLC-MS) instrument for detection, and record the chromatograms. Qualitatively analyze based on the retention times and mass numbers of the impurities in the impurity reference solutions and the test solution, and calculate the contents of the impurities by the external standard method.

[0015] The conditions for 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), select the mass-to-charge ratios of DQT, NOAC, NOND, CNO, NOEE, and NOCD to be 112.2, 534.2, 641.2, 550.2, 562.2, and 581.2.

[0016] 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).

[0017] 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.

[0018] The conditions for gradient elution are as follows:

[0019] For the first gradient elution time of 0 min, the proportion of mobile phase A is 80% - 70%, and the proportion of mobile phase B is 20% - 30%;

[0020] For the second gradient elution time of 25 min, the proportion of mobile phase A is 35% - 25%, and the proportion of mobile phase B is 65% - 75%;

[0021] For the third gradient elution time of 30 min, the proportion of mobile phase A is 35% - 25%, and the proportion of mobile phase B is 65% - 75%;

[0022] For the fourth gradient elution time of 31 min, the proportion of mobile phase A is 80% - 70%, and the proportion of mobile phase B is 20% - 30%;

[0023] For the fifth gradient elution time of 40 min, the proportion of mobile phase A is 80% - 70%, and the proportion of mobile phase B is 20% - 30%.

[0024] 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%.

[0025] The concentration of each reference substance in each impurity reference solution is 40 ng / mL - 60 ng / mL;

[0026] The concentration of relugolix in the test solution is 1.5 mg / mL to 2.5 mg / mL.

[0027] 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.

[0028] The structural formulas and chemical formulas of the respective genotoxic impurities are shown in Table 1.

[0029] Table 1 Genotoxic Impurities

[0030]

[0031] The preferred elution conditions are shown in Table 2.

[0032] Table 2 Elution Conditions

[0033]

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) The method for determining 6 genotoxic impurities in relugolix by high - performance liquid chromatography - mass spectrometry in 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.

[0036] (2) The method for determining 6 genotoxic impurities in relugolix by high - performance liquid chromatography - mass spectrometry in 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

[0037] Figure 1LC-MS chromatograms of the blank solution for the specificity test of Example 3 in positive ion mode signal channel 1 (MSD1) and positive ion mode signal channel 2 (MSD2).

[0038] Figure 2 LC-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).

[0039] 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).

[0040] 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).

[0041] 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).

[0042] 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).

[0043] 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).

[0044] 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).

[0045] 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).

[0046] 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).

[0047] Figure 11LC-MS chromatograms of the detection limit solution in Example 4 in the positive ion mode signal channel 1 (MSD1) and the positive ion mode signal channel 2 (MSD2).

[0048] Figure 12 LC-MS chromatograms of the quantitation limit solution in Example 4 in the positive ion mode signal channel 1 (MSD1) and the positive ion mode signal channel 2 (MSD2). Detailed implementation manners

[0049] The present invention will be further described below in conjunction with specific embodiments.

[0050] The relugolix raw material used in the example is sourced from: Ruiyang Pharmaceutical Co., Ltd., batch number: 24013101. Other reagents, unless otherwise specified, are commercially available conventional raw materials.

[0051] Example 1

[0052] The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography - mass spectrometry comprises the following steps:

[0053] (1) Preparation of impurity reference solution:

[0054] DQT stock solution: Take impurity DQT, weigh accurately, dissolve with acetonitrile and dilute to a solution containing 50 μg per 1 mL.

[0055] NOND stock solution: Take NOND, weigh accurately, dissolve with acetonitrile and dilute to a solution containing 50 μg per 1 mL.

[0056] NOAC stock solution: Take NOAC, weigh accurately, dissolve with acetonitrile and dilute to a solution containing 50 μg per 1 mL.

[0057] NOCD stock solution: Take NOCD, weigh accurately, dissolve with acetonitrile and dilute to a solution containing 50 μg per 1 mL.

[0058] NOEE stock solution: Take NOEE, weigh accurately, dissolve with acetonitrile and dilute to a solution containing 50 μg per 1 mL.

[0059] CNO stock solution: Take impurity CNO, weigh accurately, dissolve with acetonitrile and dilute to a solution containing 50 μg per 1 mL.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] (2) Preparation of test solution:

[0067] 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.

[0068] (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 is 10 μL - 20 μL; the detector is a single - stage quadrupole mass detector; the column temperature is 33 - 37 °C; the flow rate is 0.95 - 1.05 mL / min; the chromatographic column is packed with octadecylsilyl silica gel; mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile; gradient elution is carried out.

[0069] Method validation of the HPLC - MS determination method for the detection of genotoxic impurities in relugolix described in the present invention.

[0070] Example 2

[0071] (1) Diluent: Acetonitrile, acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75);

[0072] Blank solution: Acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75);

[0073] 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.

[0074] Reference solution: Weigh appropriate amounts of impurities DQT, NOND, NOAC, NOCD, NOEE, and CNO accurately, dissolve them in acetonitrile, and dilute to prepare a mixed stock solution with a concentration of 50 μg / mL for each impurity; measure 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.

[0075] DQT identification solution: Weigh 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; measure an appropriate amount of the DQT stock solution and dilute it with acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity identification solution with a concentration of 50 ng / mL.

[0076] NOND identification solution: Weigh an appropriate amount of NOND, dissolve it in acetonitrile, and dilute to prepare a NOND stock solution with a concentration of 50 μg / mL; measure an appropriate amount of the NOND stock solution and dilute it with acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity identification solution with a concentration of 50 ng / mL.

[0077] NOAC identification solution: Weigh an appropriate amount of NOAC, dissolve it in acetonitrile, and dilute to prepare a NOAC stock solution with a concentration of 50 μg / mL; measure an appropriate amount of the NOAC stock solution and dilute it with acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity identification solution with a concentration of 50 ng / mL.

[0078] NOCD identification solution: Weigh an appropriate amount of NOCD, dissolve it in acetonitrile, and dilute to prepare a NOCD stock solution with a concentration of 50 μg / mL; measure an appropriate amount of the NOCD stock solution and dilute it with acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity identification solution with a concentration of 50 ng / mL.

[0079] NOEE identification solution: Weigh an appropriate amount of NOEE, dissolve it in acetonitrile, and dilute to prepare a NOEE stock solution with a concentration of 50 μg / mL; measure an appropriate amount of the NOEE stock solution and dilute it with acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity identification solution with a concentration of 50 ng / mL.

[0080] CNO identification solution: Weigh 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; measure an appropriate amount of the CNO stock solution and dilute it with acetonitrile - 0.1 wt% formic acid solution (volume ratio 25:75) to prepare an impurity identification solution with a concentration of 50 ng / mL.

[0081] Reference Intermediate Solution: Accurately pipette 1 mL each of DQT stock solution, NOND stock solution, NOAC stock solution, NOCD stock solution, NOEE stock solution and CNO stock solution, transfer them into the same 100 mL volumetric flask, dilute to the mark with acetonitrile - 0.1 wt% formic acid solution (25:75), and mix well.

[0082] Spiked Test Solution: Take 20 mg of relugolix, accurately weigh it, 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 mix well.

[0083] (2) Chromatographic and Mass Spectrometric Conditions

[0084] Chromatographic column: Waters Xbridge RP18, 4.6 mm × 250 mm, 5 µm;

[0085] Column temperature: 35 °C;

[0086] Mobile phase flow rate: 1.0 mL / min;

[0087] Injection volume: 10 μL;

[0088] 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.

[0089] Use a single - stage quadrupole mass detector, and perform selected ion monitoring (SIM) in the positive ion mode of electrospray ionization (ESI). The information collected by the dual - channel is as follows:

[0090] Positive ion mode signal channel 1: Mass - to - charge ratio (m / z) is 112.2 (impurity DQT), 534.2 (NOAC), 641.2 (NOND).

[0091] Positive ion mode signal channel 2: Mass - to - charge ratio (m / z) is 550.2 (impurity CNO), 562.2 (NOEE), 581.2 (NOCD).

[0092] Detection: Inject the prepared solution into a high - performance liquid chromatography - mass spectrometry (HPLC - MS) instrument for detection, and record the chromatogram.

[0093] (3) Calculation method: Content of each impurity (ppm) = weight 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 / weight of test sample × 100%.

[0094] Example 3

[0095] (I) Specificity test:

[0096] For the method of Example 2, by injecting blank solution, various impurity localization solutions, reference substance solution, test solution, and spiked test solution containing impurities as the specificity solution, the specificity of the method was investigated. Through the specificity test, the retention time and resolution of the potential impurity under the determination conditions were determined, and it was ensured that the impurities of concern could 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 substance solution is as Figure 2 shown, Figure 2 In MSD1 (channel 1) of , the chromatographic peaks 1, 2, and 3 are DQT, NOND, and NOAC in sequence; in MSD2 (channel 2), the chromatographic peaks 4, 5, and 6 are NOCD, NOEE, and CNO in sequence.

[0097] The test solution is as Figure 3 shown, the spiked test solution is as Figure 4 shown, the DQT localization solution is as Figure 5 shown, the NOND localization solution is as Figure 6 shown, the NOAC localization solution is as Figure 7 shown, the NOCD localization solution is as Figure 8 shown, the NOEE localization solution is as Figure 9 shown, the CNO localization solution is as Figure 10 shown. From the above detections, it can be seen that the main component and adjacent impurities as well as various impurities in the spiked test solution can be effectively separated, and the specificity is good.

[0098] Table 3 Results of Specificity Test

[0099] Conclusion: The ratios of the retention times of the target impurity peaks in the spiked test solution to the corresponding target peak retention times in the reference substance solution are all within the range of 0.98 - 1.02; the resolution between the target impurity peaks in the spiked test solution is greater than 1.5; the specificity of this method is good.

[0100] Example 4

[0101] (2) Detection Limit and Quantification Limit Tests:

[0102] Take each impurity reference substance and use 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 ratio S / N ≥ 10 is used as the quantification limit concentration. The quantification limit solution is injected continuously for 6 injections, 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 quantification limit and detection limit tests are shown in Tables 4 - 5. The liquid chromatography - mass spectrometry detection limit solution is as Figure 11 shown, and the quantification limit solution is as Figure 12 shown.

[0103] Table 4 Results of the quantitative limit test

[0104] Table 5 Results of the detection limit test

[0105]

[0106] Conclusion: When the quantitative limit solution was injected repeatedly 6 times, the RSD of the retention time was less than 1.0%, the RSD of the peak area was less than 15%, and the s / n was greater than 10; the s / n of the detection limit solution was greater than 3; the quantitative limit and the detection limit were good.

[0107] Example 5

[0108] (III) Linearity and range test:

[0109] The linearity of the method was achieved by the linearity of 6 solutions with different impurity concentrations. The measured 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 of impurity DQT are shown in Table 6.

[0110] Table 6 Results of the linear relationship test of impurity DQT

[0111] The results of the linearity test of NOND are shown in Table 7.

[0112] Table 7 Results of the linear relationship test of NOND

[0113] The results of the linearity test of NOAC are shown in Table 8.

[0114] Table 8 Results of the linear relationship test of NOAC

[0115] The results of the linearity test of NOCD are shown in Table 9.

[0116] Table 9 Results of the linear relationship test of NOCD

[0117] The results of the linearity test of NOEE are shown in Table 10.

[0118] Table 10 Results of the linear relationship test of NOEE

[0119] The results of the linearity test of impurity CNO are shown in Table 11.

[0120] Table 11 Results of the linear relationship test of impurity CNO

[0121] Conclusion: The correlation coefficients of the linearity and range of the 6 genotoxic impurities of relugolix are all greater than 0.990, indicating good linear relationships. The ratios of the Y-axis intercepts to the 100% response values are all less than 30%. The external standard method with reference substances can be used for determination.

[0122] Example 6

[0123] (IV) Repeatability test:

[0124] Six spiked test solution samples were prepared in parallel, and the RSDs of the recoveries of each impurity in the spiked test solution were calculated to investigate the repeatability of the method. The results of each impurity in the repeatability test are shown in Table 12.

[0125] Table 12 Results of repeatability test

[0126]

[0127] Conclusion: The RSDs of the recoveries of impurities in the six spiked test solution samples are ≤ 10%, meeting the requirements, indicating good repeatability of the method.

[0128] Example 7

[0129] (V) Precision

[0130] Six spiked test solution samples were prepared in parallel under different dates, by different experimenters, and under different instrument conditions, and the RSDs of the recoveries of each impurity in the spiked test solution were calculated to investigate the precision of the method. The results of each impurity in the precision test are shown in Table 13.

[0131] Table 13 Results of precision test

[0132]

[0133] Conclusion: Among the twelve spiked test solution samples, the RSDs of the measured recoveries of each impurity are all less than 15%, indicating good precision of the method.

[0134] Example 8

[0135] (VI) Accuracy

[0136] The accuracy of the method was achieved through three solutions with different impurity concentrations, and the RSDs of the recoveries of each impurity in the spiked test solution were calculated. The results of the accuracy test are shown in Tables 14 - 19.

[0137] Table 14 Results of the recovery test of impurity DQT

[0138]

[0139] Table 15 Results of the recovery test of NOND

[0140]

[0141] Table 16 NOAC Recovery Test Results

[0142]

[0143] Table 17 NOCD Recovery Test Results

[0144]

[0145] Table 18 NOEE Recovery Test Results

[0146]

[0147] Table 19 Impurity CNO Recovery Test Results

[0148]

[0149] Conclusion: From the test results, it can be seen 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 the average value of the recovery rate are between 70% - 130%, and the RSD is less than 15%, indicating good method accuracy.

[0150] Example 9

[0151] (IX) Robustness Test:

[0152] 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.

[0153] Table 20 Robustness Test Results

[0154]

[0155] 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, indicating good robustness of the method.

[0156] 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 limit of quantitation is far 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 durability, and the results are stable and reliable, so it can be used for the quality control of relugolix, providing an effective guarantee for the quality of the final product, and filling the blank of detecting genotoxic impurities in relugolix by using a high performance liquid chromatography-mass spectrometer.

Claims

1. A method for the determination of 6 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 includes: using high performance liquid chromatography - mass spectrometry (HPLC - MS), with an octadecylsilyl bonded silica gel column as the filler, formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution, using a single quadrupole mass detector, and detecting in the positive ion mode of electrospray ionization; 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%.

2. The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography - mass spectrometry according to claim 1, characterized in that: The method described above includes the following steps: (1) Preparation of impurity reference solution: Precisely weigh impurities DQT, NOND, NOAC, NOCD, NOEE, and CNO respectively, dissolve them in acetonitrile and dilute to prepare each impurity reference stock solution; precisely measure each impurity reference stock solution and dilute it with acetonitrile - formic acid aqueous solution to prepare each impurity reference solution; (2) Preparation of test solution: Precisely weigh relugolix, dissolve it in acetonitrile - formic acid aqueous solution and dilute, shake well to obtain the test solution; (3) Detection: Inject the prepared impurity reference solutions and test solution into the HPLC - MS instrument for detection, and record the chromatogram; identify by 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.

3. The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 1, wherein: The conditions for 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; in the positive ion mode of electrospray ionization, select the mass - to - charge ratios of DQT, NOAC, NOND, CNO, NOEE, and NOCD to be 112.2, 534.2, 641.2, 550.2, 562.2, and 581.

2.

4. The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography-mass spectrometry according to claim 3, wherein: The chromatographic column is Waters Xbridge RP18, 4.6 mm × 250 mm, 5 µm.

5. The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography - mass spectrometry according to claim 1, wherein: The mass concentration of formic acid in the formic acid aqueous solution is 0.08% - 0.12%.

6. The method for determining 6 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%.

7. The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography - mass spectrometry according to claim 2, characterized in that: The concentration of each reference in each impurity reference solution is 40 ng / mL - 60 ng / mL.

8. The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography - mass spectrometry according to claim 2, wherein: The concentration of relugolix in the test solution is 1.5 mg / mL - 2.5 mg / mL.

9. The method for determining 6 genotoxic impurities in relugolix by high performance liquid chromatography - mass spectrometry according to claim 1, wherein: 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.

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