A method for detecting related substances in crisaborole

Abstract

The present invention relates to a method for detecting related substances in ritastatins. Appropriate chromatographic conditions and solvents for dissolving samples are screened. A gradient elution is performed using an aqueous solution of 0.01% to 0.5% phosphoric acid and acetonitrile as a mixed mobile phase. The elution time and the proportion of the mobile phase are optimized to achieve effective separation between the impurity benzyl alcohol and other impurities. Many impurities can be detected, with high sensitivity, good specificity, and high durability. The main component and each impurity have strong retention ability and high response in this detection method, and the separation between components is good. It can quickly and accurately monitor the related substances in ritastatins, which is of great significance for the quality evaluation of ritastatin raw materials and preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting related substances in lifalast. Background Art

[0002] Lifitegrast is a new type of small molecule integrin inhibitor that can antagonize lymphocyte function-associated antigen-1 (LFA-1), block the interaction with its cognate ligand intercellular adhesion molecule-1 (ICAM-1), interfere with the overexpression of ICAM-1 in corneal and conjunctival tissues that causes dry eye disease, and is the first drug to treat the symptoms and signs of dry eye disease. Lifitegrast introduces multiple impurities during the synthesis and storage process, and a method needs to be developed to quantitatively analyze the impurities, strictly control the quality of lifitegrast, and ensure that the product quality meets the requirements.

[0003] Lifalast, chemical name is (S)-2-(2-(benzofuran-6-carbonyl)-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)-3-(3-(methylsulfonyl)phenyl)propionic acid, the structural formula is as follows:

[0004]

[0005] At present, although there are reports on the detection methods of related substances in Litamilast, there are still many defects. For example, patent CN111060629A discloses a method for determining related substances in Litamilast using high performance liquid chromatography, but it can only determine six impurities, namely impurities A to F, while Litamilast introduces more than these six impurities during the synthesis and storage process, and it is obviously impossible to achieve accurate control of the quality of Litamilast.

[0006] Although patent CN114689733A reports the detection method of 14 impurities, the separation degree of adjacent impurities JP-Y-8, SM1 and JP-Y-9 is not high, and the reproducibility is relatively poor. Moreover, impurity JP-Y-12 is very close to the solvent peak, and the solvent peak is very likely to interfere with the accurate quantification of impurity JP-Y-12 during the stability test. More importantly, benzyl alcohol is not reported in the impurities reported in the patent, and there is no report on the detection method of impurity benzyl alcohol in lifalast. However, the impurity is detected in multiple batches of reference preparations. Whether the impurity benzyl alcohol can be effectively detected and quantified is crucial for the quality comparison study of lifalast API and preparation. Summary of the invention

[0007] The object of the present invention is to provide a method for detecting related substances in ritlecitinib on the basis of the prior art, which can detect more impurities, has high sensitivity and good specificity, has good resolution between impurity peaks, between ritlecitinib and its adjacent impurity peaks, and can quickly, effectively and accurately monitor the related substances in ritlecitinib raw materials and preparations.

[0008] The technical solution of the present invention is as follows:

[0009] A method for detecting related substances in ritlecitinib, which uses high performance liquid chromatography to quantitatively detect related substances in ritlecitinib. The high performance liquid chromatography conditions include: the chromatographic column is an octadecylsilane-bonded silica gel column, and gradient elution is carried out with mobile phase A and mobile phase B as the mixed mobile phase. Mobile phase A is an aqueous solution of 0.01% - 0.5% phosphoric acid, and mobile phase B is acetonitrile; the specific gradient elution process is as follows: (1) within 0 - 5 minutes, the volume ratio of mobile phase A and mobile phase B remains at 80 - 95:20 - 5; (2) within 5 - 15 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 80 - 95:20 - 5 to 70:30; (3) within 15 - 45 minutes, the volume ratio of mobile phase A and mobile phase B remains at 70:30; (4) within 45 - 50 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 70:30 to 55:45; (5) within 50 - 65 minutes, the volume ratio of mobile phase A and mobile phase B remains at 55:45; (6) within 65 - 80 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) within 80 - 85 minutes, the volume ratio of mobile phase A and mobile phase B remains at 30:70; (8) within 85 - 86 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 30:70 to 80 - 95:20 - 5; (9) within 86 - 95 minutes, the volume ratio of mobile phase A and mobile phase B remains at 80 - 95:20 - 5.

[0010] In the present invention, the test sample for the detection of related substances in ritlecitinib is ritlecitinib raw material or ritlecitinib eye drops.

[0011] For the detection method provided by the present invention, gradient elution is carried out with mobile phase A and mobile phase B as the mixed mobile phase. In chromatographic analysis, after selecting the chromatographic column, it is necessary to determine the ratio of mobile phase A and mobile phase B during the gradient elution process of the mobile phase. For the present invention, there are many impurities in the impurity spectrum of this product, especially for impurity 9 (benzyl alcohol) existing in the raw material and reference preparation. The time and ratio of the mobile phase during the gradient elution process cannot be randomly selected, but need to be determined through a large number of experiments and analyses. Otherwise, the impurities cannot be effectively separated from each other, and between the main peak and adjacent impurities, which will affect the quantitative analysis of impurities and the quality control of this product.

[0012] When determining the related substances in ritatriptan benzoate by using the detection method of the present invention, during the gradient elution process, it is necessary to strictly control the ratio of the aqueous phase (mobile phase A) to the organic phase (mobile phase B) in the initial mobile phase. When the ratio of the organic phase (mobile phase B) in the initial mobile phase is too high or too low, the impurities cannot be effectively separated from each other, and the main peak cannot be effectively separated from the adjacent impurities, making it impossible to accurately determine the content of each impurity in ritatriptan benzoate. For the present invention, the ratio of the aqueous phase (mobile phase A) to the organic phase (mobile phase B) in the initial mobile phase is controlled to be 80-95:20-5, which can be but is not limited to 80:20, 85:15, 90:10 or 95:5. Preferably, the ratio of the aqueous phase (mobile phase A) to the organic phase (mobile phase B) in the initial mobile phase is 90:10.

[0013] In a preferred embodiment, the gradient elution comprises the following steps: (1) from 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10; (2) from 5 to 15 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 90:10 to 70:30; (3) from 15 to 45 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 70:30; (4) from 45 to 50 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 70:30 to 55:45; (5) from 50 to 65 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) from 65 to 80 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) from 80 to 85 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) from 85 to 86 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 30:70 to 90:10; (9) from 86 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10.

[0014] During the gradient elution process of the present invention, the mobile phase A used is an aqueous solution of 0.01% - 0.5% phosphoric acid, and the mobile phase B is acetonitrile. With the cooperation of other conditions, the separation degree between each impurity peak, between the main peak of ritatriptan benzoate and its adjacent impurity peaks is high, so that the related substances in ritatriptan benzoate can be monitored quickly, effectively and accurately. In the present invention, the mobile phase A is an aqueous solution of 0.01% - 2% phosphoric acid, wherein the volume content of phosphoric acid can be but is not limited to 0.01%, 0.02%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, 0.15% or 0.5%. Preferably, the mobile phase A is an aqueous solution of 0.02% - 0.08% phosphoric acid; in order to obtain a better separation effect, the mobile phase A is an aqueous solution of 0.05% phosphoric acid.

[0015] In the present invention, the solvent for dissolving the sample is an acetonitrile-water mixed solution with a volume ratio of 20-40:80-60. The volume ratio of acetonitrile to water in the acetonitrile-water mixed solution can be, but is not limited to, 20:80, 25:75, 30:70, 35:65 or 40:60. To obtain better results, the solvent for dissolving the sample is an acetonitrile-water mixed solution with a volume ratio of 30:70.

[0016] In the method for detecting related substances in ritlecitinib provided by the present invention, during gradient elution, an appropriate mobile phase is screened, and the elution time and the ratio of the mobile phase are optimized, so that more impurities are detected, the main component and each impurity have strong retention ability in this detection method, high response, and good separation among components, and the related substances in ritlecitinib can be monitored quickly and accurately, which is of great significance for the quality evaluation of ritlecitinib.

[0017] For the detection method provided by the present invention, the chromatographic column used is octadecylsilane-bonded silica gel; the chromatographic column models are Inertsil ODS-3, InertSustain C18 or Thermo Hypersil Gold C18; preferably Inertsil ODS-3. In a preferred embodiment, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the filler particle size is 5 μm.

[0018] Furthermore, the detection wavelength of the detector is 212-216 nm, preferably 214 nm.

[0019] Furthermore, the flow rate of the detector is 0.9-1.1 ml / min, preferably 1.0 ml / min.

[0020] Furthermore, the column temperature is 25-35 °C, preferably 30 °C.

[0021] Furthermore, the injection volume is 5-20 μl, preferably 10 μl. The injection volume can be 5 μl, 10 μl or 20 μl.

[0022] A method for detecting related substances in ritlecitinib provided by the present invention, wherein the related substances include the following substances:

[0023]

[0024]

[0025] In a preferred embodiment, for the detection method provided by the present invention, the following solution can be prepared. When preparing the following solution, the solvent selected for dissolving the sample is: an acetonitrile-water mixed solution with a volume ratio of 30:70.

[0026] Test solution: Weigh accurately 10 mg of the sample, place it in a 20-ml volumetric flask, dissolve it with the solvent and make up to the mark, shake well to obtain the test solution.

[0027] Reference solution: Pipette accurately 1 ml of the test solution into a 100-ml volumetric flask, dilute it to the mark with the solvent, shake well to obtain the reference solution.

[0028] Stock solutions of each impurity: Weigh accurately 1 mg of each of impurity 1 - 4, impurity 6, impurity 8 - 10 respectively, place them in 10-ml volumetric flasks, dissolve them with the solvent and make up to the mark, shake well to obtain solutions containing 100 μg of each impurity per 1 ml as the stock solutions of impurity 1 - 4, impurity 6, impurity 8 - 10; Weigh accurately 1 mg of impurity 5, first dissolve it with an appropriate amount of acetonitrile, then quantitatively dilute it with the solvent to prepare a solution containing 100 μg per 1 ml as the stock solution of impurity 5; Weigh accurately 1 mg of impurity 7, first dissolve it with an appropriate amount of dimethyl sulfoxide, then quantitatively dilute it with the solvent to prepare a solution containing 100 μg per 1 ml as the stock solution of impurity 7.

[0029] Stock solution of crisaborole reference substance: Weigh accurately 10 mg of crisaborole reference substance, place it in a 100-ml volumetric flask, dissolve it with the solvent and dilute to the mark, shake well to obtain a solution containing 100 μg of crisaborole reference substance per 1 ml.

[0030] Mixed impurity solution: Pipette accurately 0.5 ml of each stock solution of impurity and 5 ml of the stock solution of crisaborole reference substance into a 100-ml volumetric flask, dissolve it with the solvent and make up to the mark, shake well to obtain a solution containing 0.5 μg of each impurity and 5 μg of crisaborole per 1 ml.

[0031] Spiked sample solution: Weigh accurately 50 mg of crisaborole sample, place it in a 100-ml volumetric flask, add 0.5 ml of each stock solution of impurity, dissolve it with the solvent and make up to the mark, shake well to obtain a solution containing 0.5 mg of crisaborole sample and 0.5 μg of each impurity per 1 ml.

[0032] The present invention screens appropriate chromatographic conditions to conduct chromatographic detection on crisaborole and the above-mentioned impurities, determines the detection method of the present invention, and verifies the specificity of the present invention through peak location tests, interference tests and degradation tests of each impurity and crisaborole.

[0033] Adopting the technical solution of the present invention, the advantages are as follows:

[0034] The detection method for related substances in lifitegrast provided by the present invention screens appropriate chromatographic conditions and solvents for dissolving samples, uses an aqueous solution of 0.01% - 0.5% phosphoric acid and acetonitrile as a mixed mobile phase for gradient elution, optimizes the elution time and the proportion of the mobile phase, realizes the effective separation between the impurity benzyl alcohol and other impurities, detects many impurities, has high sensitivity, good specificity, high durability, strong retention ability of the main component and each impurity in this detection method, high response, good separation between each component, can quickly and accurately monitor the related substances in lifitegrast, and has important significance for the quality evaluation of lifitegrast raw materials and preparations. Description of the Drawings

[0035] Figure 1 is the high-performance liquid chromatography (HPLC) chromatogram of the blank solution in Example 1;

[0036] Figure 2 is the HPLC chromatogram of the test solution in Example 1;

[0037] Figure 3 is the HPLC chromatogram of the mixed impurity solution in Example 1;

[0038] Figure 4 is the HPLC chromatogram of the sample spiked solution in Example 1;

[0039] Figure 5 is the HPLC chromatogram of the mixed impurity solution in Example 2;

[0040] Figure 6 is the HPLC chromatogram of the reference preparation (trade name: Xiidra; batch number: 21CB5) in Example 2;

[0041] Figure 7 is the HPLC chromatogram of the mixed impurity solution in Comparative Example 1;

[0042] Figure 8 is the HPLC chromatogram of the mixed impurity solution in Comparative Example 2;

[0043] Figure 9 is the HPLC chromatogram of the mixed impurity solution in Comparative Example 3 and its partial enlarged view. Detailed Description of the Invention

[0044] In order to more clearly understand the technical solution of the present invention, the present invention is further described through the following examples, but these examples do not constitute any limitation to the present invention.

[0045] Example 1

[0046] High-performance liquid chromatography conditions:

[0047] The chromatographic column was an octadecylsilyl silica gel column, with the model of Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). An aqueous solution of 0.05% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B; gradient elution was performed, the detection wavelength was 214 nm, the flow rate was 1.0 ml / min, the column temperature was 30 °C, and the injection volume was 10 μl.

[0048] The specific gradient elution process was as follows: (1) From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 90:10; (2) From 5 to 15 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed uniformly from 90:10 to 70:30; (3) From 15 to 45 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 70:30; (4) From 45 to 50 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed uniformly from 70:30 to 55:45; (5) From 50 to 65 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 55:45; (6) From 65 to 80 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed uniformly from 55:45 to 30:70; (7) From 80 to 85 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 30:70; (8) From 85 to 86 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed uniformly from 30:70 to 90:10; (9) From 86 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 90:10.

[0049] The preparation of the solutions was as follows:

[0050] The solvent for dissolving the sample was: an acetonitrile-water mixed solution with a volume ratio of 30:70, which was the blank solution.

[0051] Test solution: Accurately weigh 10 mg of the sample, place it in a 20 ml volumetric flask, dissolve it with the solvent and make up to the mark, shake well, and it is ready.

[0052] Control solution: Accurately pipette 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with the solvent, shake well, and it is ready.

[0053] Stock solutions of each impurity: Accurately weigh 1 mg of each of impurity 1 - 4, impurity 6, and impurity 8 - 10 respectively, place them in 10 ml volumetric flasks, dissolve them with the solvent and make up to the mark, shake well, to obtain solutions containing 100 μg of each impurity per 1 ml, which are used as the stock solutions of impurity 1 - 4, impurity 6, and impurity 8 - 10; accurately weigh 1 mg of impurity 5, first dissolve it with an appropriate amount of acetonitrile, and then quantitatively dilute it with the solvent to prepare a solution containing 100 μg per 1 ml, which is used as the stock solution of impurity 5; accurately weigh 1 mg of impurity 7, first dissolve it with an appropriate amount of dimethyl sulfoxide, and then quantitatively dilute it with the solvent to prepare a solution containing 100 μg per 1 ml, which is used as the stock solution of impurity 7.

[0054] Reserve solution of crisaborole reference substance: Weigh accurately 10 mg of crisaborole reference substance, place it in a 100 ml volumetric flask, dissolve it with solvent and dilute to the scale, shake well to obtain a solution containing 100 μg of crisaborole reference substance per 1 ml.

[0055] Mixed impurity solution: Pipette accurately 0.5 ml of each impurity reserve solution and 5 ml of the crisaborole reference substance reserve solution, place them in a 100 ml volumetric flask, make up the volume to the scale with solvent, shake well to obtain a solution containing 0.5 μg of each impurity and 5 μg of crisaborole per 1 ml.

[0056] Spiked sample solution: Weigh accurately 50 mg of the crisaborole sample, place it in a 100 ml volumetric flask, add 0.5 ml of each impurity reserve solution, make up the volume to the scale with solvent, shake well to obtain a solution containing 0.5 mg of the crisaborole sample and 0.5 μg of each impurity per 1 ml.

[0057] Take 10 μl of each of the above solutions, inject them for analysis, record the chromatogram, and the relevant chromatograms are shown in Figure 1 、 Figure 2 、 Figure 3 and Figure 4 。

[0058] It can be seen from Figures 1 - 4 that the baseline is stable, the peak shapes of each impurity are good, and the blank solution does not interfere with the determination of each impurity; in the chromatogram of the test solution, only impurity 5 is detected, and the detected amount (area normalization method) is 0.037%; in the spiked sample solution, the resolution between each impurity and between the main component and the adjacent impurity is good.

[0059] Verify the related substances detection method as follows:

[0060] 1. Specificity

[0061] Prepare the blank solvent, mixed impurity solution, test solution and spiked sample solution with reference to Example 1 above. Pipette accurately 10 μl of the blank solvent, mixed impurity solution, test solution and spiked sample solution respectively, inject them into the liquid chromatograph, record the chromatogram, and the relevant chromatograms are shown in Figures 1 - 4 ,and the results are shown in Table 1.

[0062] Table 1 Spiked sample solution

[0063] Name Retention Time (min) Theoretical Plate Number Resolution Tailing Factor Impurity 6 13.038 31441.09 / 0.74 Impurity 3 16.425 195112.21 15.3 1.05 Impurity 9 18.193 114418.9 9.74 1.02 Impurity 4 21.738 38829.5 10.79 0.95 Impurity 8 22.830 66742.72 2.75 0.62 Impurity 2 29.607 57994.33 16.03 1.32 Impurity 10 31.286 51845.19 3.23 1.02 Unknown 51.938 347416.28 45.8 1.17 Unknown 53.929 391781.25 5.71 1.02 Ritanserin 55.710 453794.69 5.27 0.96 Impurity 7 57.135 270026.18 3.69 1.23 Unknown 61.800 247594.89 9.97 0.93 Unknown 64.820 192060.39 5.55 1.21 Unknown 68.425 62153.81 4.27 1.03 Impurity 1 72.476 214755.56 4.70 1.43 Impurity 5 79.438 1173341.37 15.16 1

[0064] Conclusion: Under these chromatographic conditions, the baseline is stable, the blank solvent has no interference; the resolution in the mixed impurity solution is greater than 1.5; the resolution between the main component and the adjacent impurity in the spiked sample solution is 5.27 and 3.69, the theoretical plate number of the main component is 453794.69, and the resolution between each impurity is greater than 1.5, and the method has good specificity.

[0065] 2. Forced degradation test

[0066] Undamaged sample solution: Take 10 mg of ritlecitinib sample, place it in a 20-ml volumetric flask, dissolve it with the solvent and dilute to the mark, shake well, and you will get it.

[0067] Acid-damaged sample solution: Take 10 mg of ritlecitinib sample, place it in a 20-ml volumetric flask, add 1 ml of 1 M hydrochloric acid solution, let it stand at room temperature for 24 h, add 1 ml of 1 M sodium hydroxide solution to neutralize it, and then dilute to the mark with the solvent, and you will get it.

[0068] Base-damaged sample solution: Take 10 mg of ritlecitinib sample, place it in a 20-ml volumetric flask, add 1 ml of 1 M sodium hydroxide solution, let it stand at room temperature for 24 h, add 1 ml of 1 M hydrochloric acid solution to neutralize it, and then dilute to the mark with the solvent, and you will get it.

[0069] Acid-base blank solution: Accurately measure 1 ml of 1 M hydrochloric acid solution and 1 ml of sodium hydroxide solution respectively into the same 20-ml volumetric flask, add the solvent to make up the volume to the mark, shake well, and you will get it.

[0070] Oxidation-damaged sample solution: Take 10 mg of ritlecitinib sample, place it in a 20-ml volumetric flask, add 2 ml of 3% hydrogen peroxide solution, let it stand at room temperature for 24 h, and then dilute to the mark with the solvent, shake well, and you will get it. The blank solvent is prepared in the same way.

[0071] Liquid high-temperature damaged sample solution: Take 10 mg of ritlecitinib sample, place it in a 20-ml volumetric flask, dissolve it with about 10 ml of the solvent, destroy it in a water bath at 100 °C for 8 h, take it out and cool it, and then dilute to the mark with the solvent, shake well, and you will get it.

[0072] Solid high-temperature damaged sample solution: Take 10 mg of ritlecitinib sample, place it in a 20-ml volumetric flask, destroy it at 100 °C for 8 h, take it out and cool it, dissolve it with the solvent and dilute to the mark, shake well, and you will get it.

[0073] Liquid light-damaged sample solution: Take 10 mg of ritlecitinib sample, place it in a 20-ml transparent volumetric flask, dissolve it with the solvent and dilute to the mark, shake well, put it in a light box (5000 lx) for light damage for 2 days, take it out, and you will get it. The blank solvent is prepared in the same way.

[0074] Solid light-damaged sample solution: Take 10 mg of ritlecitinib sample, place it in a 20-ml transparent volumetric flask, put it in a light box (5000 lx) for light damage for 2 days, take it out, dissolve it with the solvent and dilute to the mark, shake well, and you will get it.

[0075] Accurately measure 10 μl of each damaged solution, inject it into the liquid chromatograph, and record the chromatogram.

[0076] The results showed that this product degraded to varying degrees under the conditions of acid, oxidation, high temperature and light, and the degree of degradation was the most severe under the condition of alkali. Under each degradation condition, impurity 2 was produced by degradation. Under the conditions of acid and alkali degradation, impurity 6 was also produced by degradation; under the condition of oxidation degradation, impurity 5 was also produced by degradation; under the conditions of high temperature and light degradation, both impurity 5 and impurity 6 were produced by degradation. Under various degradation conditions, the resolution between the degradation products and the main component was greater than 1.5. The peak purity of the main peak under each degradation condition was greater than 990, and the material balance was between 90% and 110%. The method specificity was good. The material balance of each degradation test is shown in Table 2.

[0077] Table 2 Investigation of Material Balance

[0078]

[0079]

[0080] 3. Detection Limit and Quantification Limit

[0081] Prepare stock solutions of rimexolone reference substance and each impurity (impurity 1 - 10) at a certain concentration, and gradually dilute them. The signal-to-noise ratios S / N≈10 and S / N≈3 are used as the quantification limit and detection limit respectively.

[0082] Determination: Take 10 μl of each of the quantification limit and detection limit solutions, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 3.

[0083] Table 3 Results of Quantification Limit and Detection Limit of Each Impurity

[0084]

[0085] It can be seen from the above table that the detection limits and quantification limits of rimexolone and each impurity meet the requirements of detection sensitivity, fully verifying the high detection sensitivity of the detection method of the present invention.

[0086] 4. Linearity and Range

[0087] (1) The stock solutions of each impurity and the main component rimexolone are the same as those under the specificity item

[0088] (2) Accurately pipette 2.5 ml of the stock solution of each impurity and 5 ml of the stock solution of the main component, place them in a 50 ml volumetric flask, add solvent to volume to the mark, shake well, and obtain a mixed impurity stock solution containing 50 μg / ml of rimexolone and 5 μg / ml of impurity per 1 ml.

[0089] (3) Dilute with solvent according to Table 4 to prepare a series of solutions at different concentration levels as the impurity linear solutions.

[0090] Table 4 Preparation Method of Each Impurity Linear Solution

[0091]

[0092]

[0093] (4) Let the injection concentration (μg / ml) be the abscissa (X-axis), and the peak area be the ordinate (Y-axis). The linear ranges and linear equations of ritlecitinib and each impurity are shown in Table 5.

[0094] Table 5 Results of linearity test

[0095]

[0096]

[0097] The results show that there is a good linear relationship between the peak areas of each impurity and the concentration. The ratios of the Y-axis intercepts of the main component and each impurity to the 100% response value are all less than 25%.

[0098] 5. Correction factor

[0099] Take ritlecitinib reference standard and reference standards of impurities 1 - 10. Prepare linear solutions according to the preparation method under linearity. Use the concentration as the abscissa and the peak area as the ordinate for linear regression and obtain the standard curves of each impurity. The ratio of the slope of the standard curve of ritlecitinib to the slope of the standard curve of each impurity is the correction factor. Determinations are carried out using different chromatographic columns and different instruments. The correction factors of each impurity are determined using the slope ratio method of the linear equation, and the results are shown in Table 6.

[0100] Table 6 Correction factors of each impurity

[0101]

[0102]

[0103] 6. Injection precision

[0104] Prepare the mixed impurity solution in the same way as in the specificity test. Take 10 μl of the mixed impurity solution and inject it continuously for 6 times into the liquid chromatograph, and record the chromatogram. The results are shown in Table 7.

[0105] Table 7 Results of injection precision

[0106]

[0107]

[0108] The results show that when the mixed impurity solution is injected continuously for 6 times, the RSD of the peak areas of ritlecitinib and each impurity is less than 10.0%, and the RSD of the retention time is less than 1.0%, indicating good injection precision.

[0109] 7. Repeatability

[0110] Take this product, accurately weigh 10 mg, place it in a 20-ml volumetric flask, dissolve it with the solvent and make up to the mark, shake well, and use it as the test solution. Prepare 6 replicates in parallel.

[0111] Accurately pipette 1 ml of the test solution, place it in a 100-ml volumetric flask, dilute it to the mark with the solvent, shake well, and use it as the control solution.

[0112] Accurately pipette 10 μl of each of the above solutions and inject them into the liquid chromatograph respectively. Calculate the content of related substances in this product by the self-control method. The results are shown in Table 8.

[0113] Table 8 Results of repeatability test

[0114] Name Impurity 5 (%) Maximum Single Impurity (%) Total Impurities (%) Sample 1 0.03 0.08 0.37 Sample 2 0.03 0.08 0.38 Sample 3 0.04 0.08 0.38 Sample 4 0.04 0.08 0.38 Sample 5 0.04 0.08 0.38 Sample 6 0.03 0.08 0.38 Average Value (%) 0.04 0.08 0.38 RSD (%) 3.74 0 1.08

[0115] Conclusion: For the 6 test solutions of this product, the RSDs of the maximum single impurity and total impurity detections are both less than 10%, the impurity detection situations are basically the same, and the repeatability is good.

[0116] 8. Intermediate precision

[0117] To investigate the dispersion degree of the results obtained from a series of detections of the same homogeneous test sample by different analysts through multiple samplings at different times and on different instruments under this test condition, we conducted an intermediate precision test. The solution preparation method is the same as that of the repeatability test, and the determination results are shown in Table 9.

[0118] Table 9 Results of intermediate precision test

[0119]

[0120]

[0121] The results show that the impurity detection situations of the 12 samples of this product are the same, the RSDs (%) of the maximum single impurity and total impurity detections are both less than 10.0%, and the intermediate precision is good.

[0122] 9. Accuracy

[0123] Matrix solution: Accurately weigh 10 mg of the sample, place it in a 20-ml volumetric flask, dissolve it with the solvent and make up to the mark, shake well, and obtain it.

[0124] Reference stock solution: The same as the specific mixed impurity stock solution 1.

[0125] Reference solution: Accurately pipette 1 ml of the reference stock solution, place it in a 10-ml volumetric flask, dilute it to the mark with the solvent, shake well, and obtain it.

[0126] Low-concentration (80%) recovery: Weigh accurately 12.5 mg of the sample, place it in a 25-ml volumetric flask, add 2 ml of the reference stock solution, add the solvent, sonicate to dissolve, and dilute to the mark. Shake well to obtain the solution. Prepare three parallel samples.

[0127] Medium-concentration (100%) recovery: Weigh accurately 12.5 mg of the sample, place it in a 25-ml volumetric flask, add 2.5 ml of the reference stock solution, add the solvent, sonicate to dissolve, and dilute to the mark. Shake well to obtain the solution. Prepare three parallel samples.

[0128] High-concentration (120%) recovery: Weigh accurately 12.5 mg of the sample, place it in a 25-ml volumetric flask, add 3 ml of the reference stock solution, add the solvent, sonicate to dissolve, and dilute to the mark. Shake well to obtain the solution. Prepare three parallel samples.

[0129] Precisely measure 10 μl of each of the above solutions, inject them into the liquid chromatograph, perform the recovery determination, and calculate the recovery rate by the external standard method. The results are shown in Table 11.

[0130] Table 11 Results of the accuracy test

[0131]

[0132]

[0133] The results show that the limit of impurities is 0.1%, the recovery rates of each impurity are within the range of 90% - 110%, and the RSD (%) is less than 10.0%. The accuracy of this method is good.

[0134] 10. Solution stability

[0135] The preparation of the test solution and the reference solution is described in the repeatability section, and the preparation of the mixed impurity solution is described in the specificity section. Take the above solutions and inject them for analysis after standing at room temperature for different times to investigate the stability of each solution.

[0136] The results show that the control solution and the test solution have good solution stability when standing at room temperature for 64 hours; the mixed impurity solution has good solution stability when standing at room temperature for 68 hours.

[0137] Example 2

[0138] High-performance liquid chromatography conditions:

[0139] The chromatographic column is an octadecylsilane-bonded silica gel column, model Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). The mobile phase A is 0.05% phosphoric acid aqueous solution, and the mobile phase B is acetonitrile; gradient elution, the detection wavelength is 214 nm, the column temperature is 30 °C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0140] The specific gradient elution process is as follows: (1) From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10; (2) From 5 to 15 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 90:10 to 70:30; (3) From 15 to 45 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 70:30; (4) From 45 to 50 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 70:30 to 55:45; (5) From 50 to 65 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) From 65 to 80 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) From 80 to 85 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) From 85 to 86 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 30:70 to 90:10; (9) From 86 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10.

[0141] The preparation of the solution is as follows:

[0142] The solvent is: an acetonitrile - water mixed solution with a volume ratio of 30:70.

[0143] Test solution: Take the reference preparation of loteprednol etabonate eye drops (trade name: Xiidra; batch number: 21CB5), and dilute it with the solvent to prepare a solution containing 0.5 mg of loteprednol etabonate per 1 ml.

[0144] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 - ml volumetric flask, dilute it to the mark with the solvent, and shake well. The resulting solution contains 5 μg of loteprednol etabonate per 1 ml.

[0145] The preparation of the mixed impurity solution: The same as in Example 1.

[0146] Take 10 μl each of the test solution, the control solution, and the mixed impurity solution, inject them for analysis, record the chromatogram, and the relevant chromatograms are shown in Figure 5 and Figure 6 .

[0147] From Figure 5 and Figure 6 it can be seen that impurity 9 is detected in the reference preparation (trade name: Xiidra; batch number: 21CB5).

[0148] As for the reference preparations (trade name: Xiidra, batch numbers 20S22 and 22B23), they are detected under the same conditions as in Example 2. Similar to Example 2, impurity 9 is detected in all of them.

[0149] Example 3

[0150] High - performance liquid chromatography conditions:

[0151] The chromatographic column is an octadecylsilyl-bonded silica gel column with the model of Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). The mobile phase A is an aqueous solution of 0.05% phosphoric acid, and the mobile phase B is acetonitrile; gradient elution is performed. The detection wavelengths are 212 nm and 216 nm respectively, the column temperature is 30 °C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0152] The specific gradient elution process is as follows: (1) From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10; (2) From 5 to 15 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 90:10 to 70:30; (3) From 15 to 45 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 70:30; (4) From 45 to 50 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 70:30 to 55:45; (5) From 50 to 65 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) From 65 to 80 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) From 80 to 85 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) From 85 to 86 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 30:70 to 90:10; (9) From 86 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10.

[0153] Preparation of the mixed impurity solution and the test solution: The same as in Example 1.

[0154] Take 10 μl each of the mixed impurity solution and the test solution, inject for analysis, and record the chromatogram.

[0155] In this example, the wavelengths are adjusted to 212 nm and 216 nm to examine the resolution between the ritastat peak and the adjacent impurity peaks and among the various impurities, as well as the impurity detection in the sample. The results show that the resolution between the components is good, and the detection effect is the same as that in Example 1.

[0156] In summary, within the wavelength range of 212 nm to 226 nm, the detection effect is good.

[0157] Example 4

[0158] High performance liquid chromatography conditions:

[0159] The chromatographic column was an octadecylsilyl-bonded silica gel column, with the model of Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). 0.05% phosphoric acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B; gradient elution was performed. The detection wavelength was 214 nm, the column temperature was 25 °C and 35 °C respectively, the flow rate was 1.0 ml / min, and the injection volume was 10 μl.

[0160] The specific gradient elution process was as follows: (1) From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 90:10; (2) From 5 to 15 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed uniformly from 90:10 to 70:30; (3) From 15 to 45 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 70:30; (4) From 45 to 50 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed uniformly from 70:30 to 55:45; (5) From 50 to 65 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 55:45; (6) From 65 to 80 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed uniformly from 55:45 to 30:70; (7) From 80 to 85 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 30:70; (8) From 85 to 86 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed uniformly from 30:70 to 90:10; (9) From 86 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remained unchanged at 90:10.

[0161] Preparation of the mixed impurity solution and the test solution: The same as in Example 1.

[0162] Take 10 μl each of the mixed impurity solution and the test solution, inject for analysis, and record the chromatogram.

[0163] In this example, the column temperature was adjusted to 25 °C and 35 °C to investigate the resolution between the ritastatine peak and the adjacent impurity peaks and among the various impurities, as well as the impurity detection in the sample. The results showed that the resolution between the components was good, and the detection effect was the same as in Example 1.

[0164] In summary, within the range of 25 °C to 35 °C for the column temperature, the detection effect was good.

[0165] Example 5

[0166] High performance liquid chromatography conditions:

[0167] The chromatographic column was an octadecylsilyl-bonded silica gel column, with the model of Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). 0.05% phosphoric acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B; gradient elution was performed. The detection wavelength was 214 nm, the column temperature was 30 °C, the flow rates were 0.9 ml / min and 1.1 ml / min respectively, and the injection volume was 10 μl.

[0168] The specific gradient elution process is as follows: (1) From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10; (2) From 5 to 15 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 90:10 to 70:30; (3) From 15 to 45 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 70:30; (4) From 45 to 50 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 70:30 to 55:45; (5) From 50 to 65 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) From 65 to 80 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) From 80 to 85 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) From 85 to 86 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 30:70 to 90:10; (9) From 86 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10.

[0169] Preparation of the mixed impurity solution and the test solution: The same as in Example 1.

[0170] Take 10 μl each of the mixed impurity solution and the test solution, inject them for analysis, and record the chromatogram.

[0171] In this example, the flow rate was adjusted to 0.9 ml / min and 1.1 ml / min to examine the resolution between the rimexolone peak and the adjacent impurity peaks and among the various impurities, as well as the impurity detection in the sample. The results showed that the resolution between the components was good, and the detection effect was the same as in Example 1.

[0172] In summary, when the flow rate is in the range of 0.9 ml / min to 1.1 ml / min, the detection effect is good.

[0173] Example 6

[0174] High performance liquid chromatography conditions:

[0175] The chromatographic column is an octadecylsilane-bonded silica gel column, model Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). 0.05% phosphoric acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B; gradient elution, the detection wavelength is 214 nm, the column temperature is 30 °C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0176] The specific gradient elution process is as follows: (1) From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 88:12 (or 92:8); (2) From 5 to 15 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 88:12 (or 92:8) to 70:30; (3) From 15 to 45 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 70:30; (4) From 45 to 50 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 70:30 to 55:45; (5) From 50 to 65 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) From 65 to 80 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) From 80 to 85 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) From 85 to 86 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 30:70 to 88:12 (or 92:8); (9) From 86 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 88:12 (or 92:8).

[0177] Preparation of the mixed impurity solution and the test solution: The same as in Example 1.

[0178] Take 10 μl each of the impurity solution and the test solution, inject them for analysis, and record the chromatogram.

[0179] In this example, the volume ratio of mobile phase A to mobile phase B at the starting ratio of the gradient elution program is adjusted from 90:10 to 88:12 or 92:8, and the termination ratio of the elution program is adjusted accordingly. The resolution between the ritastine peak and the adjacent impurity peaks and between various impurities, as well as the impurity detection in the sample, are investigated. The results show that the resolution between each component is good, and the detection effect is the same as in Example 1.

[0180] Example 7

[0181] High performance liquid chromatography conditions:

[0182] The chromatographic column is an octadecylsilane-bonded silica gel column, with the model Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). 0.04% phosphoric acid aqueous solution and 0.06% phosphoric acid aqueous solution are used as mobile phase A respectively, and acetonitrile is used as mobile phase B; gradient elution, the detection wavelength is 214 nm, the column temperature is 30 °C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0183] The specific gradient elution process is as follows: (1) From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10; (2) From 5 to 15 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 90:10 to 70:30; (3) From 15 to 45 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 70:30; (4) From 45 to 50 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 70:30 to 55:45; (5) From 50 to 65 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) From 65 to 80 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) From 80 to 85 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) From 85 to 86 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 30:70 to 90:10; (9) From 86 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 90:10.

[0184] Preparation of the mixed impurity solution and the test solution: The same as in Example 1.

[0185] Take 10 μl each of the impurity solution and the test solution, inject them for analysis, and record the chromatogram.

[0186] In this example, the volume ratio of phosphoric acid in mobile phase A was adjusted to 0.04% and 0.06% to investigate the resolution between the ritastine peak and the adjacent impurity peaks and among various impurities, as well as the impurity detection in the sample. The results showed that the resolution between the components was good, and the detection effect was the same as in Example 1.

[0187] In summary, when the volume ratio of phosphoric acid in mobile phase A is adjusted within the range of 0.04% - 0.06%, the detection effect is good.

[0188] Comparative Example 1

[0189] High-performance liquid chromatography conditions:

[0190] The chromatographic column is an octadecylsilane-bonded silica gel column, model Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). The mobile phase A is an aqueous solution of 0.05% phosphoric acid, and the mobile phase B is acetonitrile; gradient elution, the detection wavelength is 214 nm, the flow rate is 1.0 ml / min, the column temperature is 30 °C, and the injection volume is 10 μl.

[0191] The specific gradient elution process is as follows: (1) From 0 to 10 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 88:12; (2) From 10 to 20 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 88:12 to 68:32; (3) From 20 to 40 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 68:32; (4) From 40 to 45 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 68:32 to 55:45; (5) From 45 to 75 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) From 75 to 85 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) From 85 to 90 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) From 90 to 91 minutes, the volume ratio of mobile phase A to mobile phase B changes from 30:70 to 88:12; (9) From 91 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 88:12.

[0192] The preparation of the solution is as follows:

[0193] The preparation of the mixed impurity solution: The same as in Example 1.

[0194] Take 10 μl of the above mixed impurity solution, inject it for analysis, record the chromatogram, and the relevant chromatograms are shown in Figure 7 .

[0195] It can be seen from Figure 7 that when the elution program is changed, the solvent peak interferes with impurity 6, and impurities 3 and 9 completely overlap, affecting the accurate quantification of impurity 9.

[0196] Comparative Example 2

[0197] High performance liquid chromatography conditions:

[0198] The chromatographic column is an octadecylsilane-bonded silica gel column, with the model of Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). 0.05% phosphoric acid aqueous solution is used as mobile phase A, and 0.05% phosphoric acid aqueous solution - acetonitrile is used as mobile phase B; gradient elution, the detection wavelength is 214 nm, the flow rate is 1.0 ml / min, the column temperature is 30 °C, and the injection volume is 10 μl.

[0199] The specific gradient elution process is as follows: (1) From 0 to 10 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 88:12; (2) From 10 to 20 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 88:12 to 68:32; (3) From 20 to 40 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 68:32; (4) From 40 to 45 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 68:32 to 55:45; (5) From 45 to 75 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) From 75 to 85 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) From 85 to 90 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) From 90 to 91 minutes, the volume ratio of mobile phase A to mobile phase B changes from 30:70 to 88:12; (9) From 91 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 88:12.

[0200] The preparation of the solution is as follows:

[0201] The preparation of the mixed impurity solution: The same as in Example 1.

[0202] Take 10 μl of the above mixed impurity solution, inject it for analysis, record the chromatogram, and the relevant chromatograms are shown in Figure 8 .

[0203] It can be seen from Figure 8 that when mobile phase B is adjusted from acetonitrile to 0.05% phosphoric acid aqueous solution - acetonitrile, impurity 4 and impurity 8 are not baseline separated, which affects the accurate quantification of impurity 4 and impurity 8.

[0204] Comparative Example 3

[0205] High performance liquid chromatography conditions:

[0206] The chromatographic column is an octadecylsilane bonded silica gel column, with the model of Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm). 0.05% phosphoric acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B; gradient elution, the detection wavelength is 214 nm, the flow rate is 1.0 ml / min, the column temperature is 30 °C, and the injection volume is 10 μl.

[0207] The specific gradient elution process is as follows: (1) From 0 to 10 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 88:12; (2) From 10 to 20 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 88:12 to 68:32; (3) From 20 to 40 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 68:32; (4) From 40 to 45 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 68:32 to 55:45; (5) From 45 to 75 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 55:45; (6) From 75 to 85 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) From 85 to 90 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; (8) From 90 to 91 minutes, the volume ratio of mobile phase A to mobile phase B changes from 30:70 to 88:12; (9) From 91 to 95 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 88:12.

[0208] The solvent for dissolving the sample is: an acetonitrile-water mixed solution with a volume ratio of 50:50.

[0209] The solution is prepared as follows:

[0210] Preparation of the mixed impurity solution: The same as in Comparative Example 1.

[0211] Take 10 μl of the above mixed impurity solution, inject it for analysis, record the chromatogram, and the relevant chromatograms are shown in Figure 9 .

[0212] It can be seen from Figure 9 that when the solvent for dissolving the sample is adjusted to an acetonitrile-water mixed solution with a volume ratio of 50:50, the peak shape of impurity 6 bifurcates, affecting the accuracy of impurity 6. This solvent ratio is not appropriate. According to the experimental results, when the proportion of acetonitrile in the solvent is higher than 30%, impurity 6 will bifurcate due to the solvent effect, affecting its accurate quantification; when the proportion of acetonitrile in the solvent is lower than 30%, it is impossible to completely dissolve ralitazone.

[0213] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting related substances in ritanserin, characterized in that, the detection method uses high performance liquid chromatography for quantitative detection of related substances in ritanserin, and the high performance liquid chromatography conditions include: the chromatographic column is an octadecylsilane bonded silica column, gradient elution is carried out with mobile phase A and mobile phase B as the mixed mobile phase, the mobile phase A is an aqueous solution of 0.01% - 0.5% phosphoric acid, and the mobile phase B is acetonitrile; the specific gradient elution process is as follows: (1) within 0 - 5 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at 90:10; (2) within 5 - 15 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 90:10 to 70:30; (3) within 15 - 45 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at 70:30; (4) within 45 - 50 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 70:30 to 55:45; (5) within 50 - 65 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at 55:45; (6) within 65 - 80 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 55:45 to 30:70; (7) within 80 - 85 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at 30:70; (8) within 85 - 86 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 30:70 to 90:10; (9) within 86 - 95 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at 90:10; the related substances are as follows:

2. The method for detecting related substances in ritanserin according to claim 1, characterized in that, the solvent for dissolving the sample is an acetonitrile - water mixed solution with a volume ratio of 20 - 40:80 - 60.

3. The method for detecting related substances in ritanserin according to claim 2, characterized in that, the solvent for dissolving the sample is an acetonitrile - water mixed solution with a volume ratio of 30:

70.

4. The method for detecting related substances in ritanserin according to claim 1, characterized in that, the mobile phase A is an aqueous solution of 0.02% - 0.08% phosphoric acid.

5. The method for detecting related substances in ritanserin according to claim 4, characterized in that, the mobile phase A is an aqueous solution of 0.05% phosphoric acid.

6. The method for detecting related substances in ritanserin according to claim 1, characterized in that, the model of the chromatographic column is Inertsil ODS - 3, InertSustain C18 or Thermo Hypersil Gold C18.

7. The method for detecting related substances in ritanserin according to claim 6, characterized in that, the model of the chromatographic column is Inertsil ODS - 3.

8. The method for detecting related substances in ritanserin according to claim 7, characterized in that, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the packing particle size is 5 μm.

9. The method for detecting related substances in ritanserin according to claim 1, characterized in that, The chromatographic conditions include: the detection wavelength is 212 - 216 nm.

10. The method for detecting related substances in ritlecitinib according to claim 9, wherein, the chromatographic conditions include: the detection wavelength is 214 nm.

11. The method for detecting related substances in ritlecitinib according to claim 1, wherein, the chromatographic conditions include: the column temperature is 25 - 35 °C.

12. The method for detecting related substances in ritlecitinib according to claim 11, wherein, the chromatographic conditions include: the column temperature is 30 °C.

13. The method for detecting related substances in ritlecitinib according to claim 1, wherein, the chromatographic conditions include: the flow rate is 0.9 - 1.1 mL / min.

14. The method for detecting related substances in ritlecitinib according to claim 13, wherein, the chromatographic conditions include: the flow rate is 1.0 mL / min.

15. The method for detecting related substances in ritlecitinib according to claim 1, wherein, the chromatographic conditions include: the injection volume is 5 - 20 μl.

16. The method for detecting related substances in ritlecitinib according to claim 15, wherein, the chromatographic conditions include: the injection volume is 10 μl.

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