Method for detecting related substances in trifarotene raw material medicine

Abstract

The invention discloses a method for detecting related substances in a trifarotene bulk drug, which comprises the following steps of: carrying out gradient elution on a to-be-detected trifarotene bulk drug with a preset concentration through a mixed mobile phase of a mobile phase A and a mobile phase B by virtue of high performance liquid chromatography, and measuring quantitation limit solution chromatograms of trifarotene and various impurities; the mobile phase A is a formic acid aqueous solution with the volume ratio of 0.05-1; the mobile phase B is a formic acid acetonitrile solution with the volume ratio of 0.05-1; the initial ratio of the mobile phase A to the mobile phase B in the elution process is (88-92): (12-8), and the related substances in the trifarotene bulk drug can be effectively detected after gradient elution.

Description

Technical Field

[0001] The present application belongs to the technical field of drug analysis, and specifically relates to a method for detecting related substances in a trefarotene raw material drug, wherein nine related substances are quantitatively analyzed by high performance liquid chromatography using the detection method. Background Art

[0002] Trifarotene, chemical name: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidin-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid, molecular formula: C 29 H 33 NO 4 , molecular weight is 459.59, and its chemical structure is as follows:

[0003] Trifarotene was developed by GALDERMA, a French company, and was approved by the FDA for marketing in the United States in October 2019. The drug belongs to the fourth generation of retinoids, which can highly selectively activate the retinoic acid receptor γ (RAR-γ) in the skin, thereby reducing the impact on other retinoic acid receptor subtypes. This selective binding property helps to significantly reduce the common skin irritation reactions of traditional retinoids and improve patients' tolerance and treatment compliance.

[0004] Related substances are starting materials, intermediates, by-products and degradation impurities introduced during the synthesis and production of trefarotene. The detection of related substances can control the quality and safety of the drug. The study of related substances is one of the key projects in drug quality research, and its content is a direct indicator of the purity of the drug.

[0005] In the prior art, Chinese patent CN117451892A discloses a method for detecting related substances in a low-concentration trefarotene cream, which has a running time of 70 minutes, a long analysis time, and no research on possible organic impurities. In addition, Chinese patent CN118583981A discloses a method for determining the content of genotoxic impurities in trefarotene bulk drugs, which well controls the genotoxic impurities in the bulk drugs. However, there is no report on the method for detecting related substances in trefarotene bulk drugs. There is no method for determining related substances in trefarotene bulk drugs with low detection limit, quantitative limit, high sensitivity, strong specificity, and high precision and accuracy, especially no method for quantitatively detecting substances in trefarotene bulk drugs. Summary of the invention

[0006] The purpose of the present invention is to provide a method for detecting related substances in trefarotene raw materials, and to detect nine potential related substances in trefarotene raw materials at one time by high performance liquid chromatography. The technical problem to be solved is to provide a method for detecting related substances in trefarotene raw materials with low detection limit and quantitative limit, high sensitivity, strong specificity, high precision and high accuracy.

[0007] Another advantage of the present invention is to provide a method for detecting related substances in trefarotene bulk drug, characterized in that the method for detecting related substances in trefarotene bulk drug comprises: By means of high performance liquid chromatography, a predetermined concentration of the to-be-detected trefarotene raw material drug is gradient eluted through a mixed mobile phase of mobile phase A and mobile phase B, and a chromatogram of the quantitative limit of trefarotene and each impurity solution is measured; the mobile phase A is a formic acid aqueous solution with a volume ratio of 0.05 to 1; the mobile phase B is a formic acid acetonitrile solution with a volume ratio of 0.05 to 1; the initial ratio of mobile phase A to mobile phase B during the elution process is 88 to 92:12 to 8; The specific gradient elution process is as follows: within 0 to 12 minutes, the ratio of mobile phase A to mobile phase B is gradually changed from the initial ratio to 50 to 60:40 to 50 at a uniform speed; Within 12 to 20 minutes, the ratio of mobile phase A to mobile phase B is gradually changed from 50 to 60:40 to 50 to 40 to 50:50 to 60 at a uniform rate; Within 20 to 30 minutes, the ratio of mobile phase A to mobile phase B gradually changes from 40 to 50:50 to 60 to 1 to 10:90 to 99 at a constant speed; Within 30 to 35 minutes, the ratio of mobile phase A to mobile phase B remains constant at 1 to 10:90 to 99; In 35-36 minutes, the ratio of mobile phase A to mobile phase B was gradually changed from 1-10:90-99 to the initial ratio; During 36 to 45 minutes, the ratio of mobile phase A to mobile phase B remained unchanged from the initial ratio; The substance content in trefarotene raw material was obtained by external standard method, and the remaining unknown impurities were obtained by area normalization method.

[0008] According to an embodiment of the present application, during the gradient elution process, the initial ratio of mobile phase A to mobile phase B is 85-95:5-15.

[0009] According to one embodiment of the present application, 1 mL of formic acid is transferred into 1000 mL of water, shaken well, and ultrasonically degassed to obtain the mobile phase A.

[0010] According to one embodiment of the present application, 1 mL of formic acid is transferred into 1000 mL of acetonitrile, shaken well, and ultrasonically degassed to obtain the mobile phase B.

[0011] According to an embodiment of the present application, the HPLC conditions at least meet at least one of the following conditions: The length of the chromatographic column in the high performance liquid chromatography method used is 50 mm to 250 mm; The inner diameter of the chromatographic column is 3 mm to 6 mm; The filler particle size of the chromatographic column is 2 μm to 6 μm; The flow rate of the chromatographic column is 0.5 to 2 mL / min; The column temperature of the chromatographic column is 25-35°C; The detection wavelength of the chromatographic column is 190-400 nm; The injection volume of the chromatographic column is 5 to 10 μL.

[0012] According to one embodiment of the present application, the HPLC conditions include: a flow rate of 1.2 mL / min; a column temperature of 30° C.; a detection wavelength of 296 nm; and an injection volume of 5 μL.

[0013] According to one embodiment of the present application, the related substances include Rtd-002-IMP1: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(1H-pyrrol-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid; Rtd-002-IMP2: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidin-1-yl)-[1,1':3',1'-triphenyl]-4-carbonitrile; Rtd-002-IMP3: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidin-1-yl)-[1,1':3',1'-triphenyl]-4-carboxamide; Rtd-002-IMP4: 4'-(2-hydroxyethoxy)-[1,1'-biphenyl]-4-carboxylic acid; Rtd-002-IMP5: 4'-(2-hydroxyethoxy)-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid; Rtd-002-INT2: 4'-hydroxy-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid ethyl ester; Rtd-002-A: 4'-(2-acetoxyethoxy)-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid ethyl ester; Rtd-002-B: 4'-(2-acetoxyethoxy)-3'-(tert-butyl)-4'-(pyrrolidin-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid ethyl ester; Rtd-002-B-IMP2: 3''(tert-butyl)-4'-(2-hydroxyethoxy)-4''(pyrrolidin-1-yl)-[1,1':3',1''-triphenyl]-4-carboxylic acid ethyl ester; at least one of the following. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the resolution solution chromatogram; Figure 2 is the chromatogram of the test solution; Figure 3 is the solution chromatogram of trefarotene and the quantitative limit of each impurity; Figure 4 This is the linear relationship diagram of trefarotene and its impurities. DETAILED DESCRIPTION

[0015] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0016] In a preferred embodiment, the high performance liquid chromatography detection method for related substances in the trefarotene bulk drug provided by the present invention preferably comprises the following steps: (1) Solution preparation Take an appropriate amount of trefarotene sample, weigh it accurately, dissolve it in methanol and dilute it quantitatively to make a solution containing about 250 μg of trefarotene per 1 mL as the test solution. Accurately weigh an appropriate amount of each impurity reference substance, dissolve it in solvent and dilute it quantitatively to make a mixed solution containing about 0.25 μg of each impurity reference substance per 1 mL as the impurity reference substance solution.

[0017] (2) Inject the test solution and the impurity reference solution into the liquid chromatograph respectively, record the chromatogram, and calculate the peak area according to the external standard method. Preferably, the chromatographic conditions are as follows: The chromatographic column is Waters XBridge BEH C18 4.6*150mm, 3.5μm.

[0018] The detection wavelength is 190-400 nm, preferably 296 nm.

[0019] The flow rate is 1.1-1.3 mL / min, preferably 1.2 mL / min.

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

[0021] The injection volume is 5 to 10 μL, preferably 5 μL.

[0022] Mobile phase A is a 0.1% by volume formic acid aqueous solution, which can be obtained by transferring 1 mL of formic acid into 1000 mL of water, shaking well, and ultrasonically degassing.

[0023] Mobile phase B is a 0.1% by volume formic acid acetonitrile solution, which can be obtained by pipetting 1 mL of formic acid into 1000 mL of acetonitrile, shaking well, and ultrasonically degassing.

[0024] Perform gradient elution according to Table 1: Table 1

[0025] It is worth mentioning that the detection method of related substances in trefarotene bulk drug provided by the present invention can detect nine potential related substances in trefarotene bulk drug at one time during the high performance liquid chromatography analysis process. The separation between each impurity and between the impurity and the main component is good, and the response of each component is high. The detection limit and quantitative limit are low, the sensitivity is high, the specificity is strong, and the precision and accuracy are high. The method can quickly and easily detect and accurately quantify the related substances in trefarotene bulk drug, greatly improving the efficiency and accuracy of drug analysis work, and providing strong technical support for the quality control of trefarotene bulk drug.

[0026] As an example, when selecting drugs and reagents, the following are selected: trefarotene (Rtd-002) reference substance, preferably, it can be made by yourself, with a content of 98.99%); Rtd-002-IMP1 reference substance, preferably, it can be made by yourself, with a purity of 81.87%; Rtd-002-IMP2 reference substance, preferably, it can be made by yourself, with a purity of 97.76%; Rtd-002-IMP3 reference substance, preferably, it can be made by yourself, with a purity of 98.71%; Rtd-002-IMP4 reference substance, preferably, it can be made by yourself, with a purity of 99.46%; Rtd-002-IMP5 reference substance, preferably, it can be made by yourself, with a purity of 94.81%); Rtd-002-INT2 reference substance, preferably, it can be made by yourself, with a purity of 96.92%)); Rtd-002-A reference substance, preferably, can be made by yourself, with a purity of 94.51%); Rtd-002-B reference substance, preferably, can be made by yourself, with a purity of 96.94%); Rtd-002-B-IMP2 reference substance, preferably, can be made by yourself, with a purity of 92.61%); Sodium chloride, preferably with specifications: analytical grade, 3A Chemical; Hydrochloric acid, preferably with specifications: analytical grade, Sinopharm Chemical Reagent Co., Ltd.; Sodium hydroxide, preferably with specifications: analytical grade, Shanghai Boer Chemical Reagent Co., Ltd.; Formic acid, preferably: chromatographic grade, damas-beta; Methanol, preferably: chromatographic grade, damas-beta; Acetonitrile, preferably: chromatographic grade, damas-beta; Ultrapure water, preferably Watsons. In addition, when selecting instruments, it is preferred to select: Agilent 1260 high performance liquid chromatograph, Shimadzu AUW220D electronic balance, electric constant temperature drying oven.

[0027] More preferably, when selecting liquid chromatography conditions, the chromatographic column uses Waters XBridge BEH C18 4.6*150mm, 3.5μm, with a 0.1% by volume formic acid aqueous solution as mobile phase A, and a 0.1% by volume formic acid acetonitrile solution as mobile phase B, gradient elution is performed according to Table 2, the detection wavelength is 296nm, the flow rate is 1.2mL / min, the column temperature is 30°C, 5μL of the test solution and the impurity reference solution are accurately measured, respectively injected into the liquid chromatograph, and the chromatogram is recorded.

[0028] Table 2

[0029] When detecting related substances in trefarotene, first take about 25 mg of trefarotene, preferably a sample with batch number 20240705, accurately weigh it, place it in a 100 mL volumetric flask, add methanol to dissolve it and dilute it to the scale, shake it well, and use it as the test solution. Accurately weigh an appropriate amount of each impurity reference substance, add a solvent to dissolve it and quantitatively dilute it to make a mixed solution containing about 0.25 μg of each impurity reference substance per 1 mL, as the impurity reference solution. Accurately measure 5 μL of the test solution and the impurity reference solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. The known impurities are quantified by the external standard method, and the unknown impurities are determined by the area normalization method. The test results are shown in Table 3 below: Table 3

[0030] 1. Verify through the following examples.

[0031] Example 1: Specificity (1) Blank solvent: methanol; (2) Test solution: Take about 25 mg of trefarotene, preferably batch number 20240705, accurately weigh it, place it in a 100 mL volumetric flask, add methanol to dissolve it and dilute it to the mark, and shake well.

[0032] (3) Separation solution: Weigh appropriate amounts of trefoil reference substance (preferably batch number 20240717) and each impurity reference substance, dissolve in solvent and quantitatively dilute to make a mixed solution containing approximately 0.25 μg of each impurity and 250 μg of trefoil per 1 mL, and shake well.

[0033] (4) Single impurity location solution: Take an appropriate amount of each impurity reference substance, dissolve it in solvent and dilute it into a solution of a certain concentration, and shake well.

[0034] Accurately measure 5 μL of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in Table 4, and the relevant spectra are shown in the attached Figure 1 and Figure 2 .

[0035] Table 4 Specificity test results

[0036] The results showed that under the chromatographic conditions, the baseline was stable and the blank solvent had no interference with the determination of this product. In the separation solution, the peaks of each substance were good, the theoretical plate number of the main peak of trefarotene was 120426.57, the separation between the main peak and the adjacent peak was 1.63, ≥1.5, and the separation between each impurity was good. The retention time ratios in the separation solution and the positioning solution of each impurity were between 0.98 and 1.02, indicating good specificity.

[0037] Example 2: Destruction Test In order to investigate whether the possible degradation products of trefarotene can be detected under the selected chromatographic conditions, the product was destroyed under severe conditions such as high temperature, high humidity, acid, alkali and oxidation, and the destroyed samples were dissolved in methanol to prepare test solutions. 5 μL of each of the above test solutions were accurately measured and injected into the liquid chromatograph to record the chromatogram. The specific methods are shown in Tables 5 and 6.

[0038] Table 5 Destruction test results

[0039] Mass balance*=(main component content after destruction + impurity content after destruction) / (main component content before destruction + total impurities before destruction).

[0040] Table 6 Impurities in the test solution under various destruction conditions

[0041] Total impurities* = the sum of all single impurities with a content ≥ 0.01%.

[0042] The results show that the product has different degrees of degradation under acid, alkali and oxidation conditions. Under oxidation conditions, the damage is the greatest, followed by alkali damage, and it is relatively stable under high temperature, high temperature and acid conditions. The degradation products produced under various damage conditions can be detected, the separation between the main component and the adjacent impurities is greater than 1.5, the separation between each impurity is greater than 1.0, the mass balance is within the range of 0.95-1.05, the main peak purity is above 0.990, and the material is conserved.

[0043] Example 3: Determination of limit of quantitation Take appropriate amount of trefarotene reference substance and each impurity reference substance, prepare a sample solution containing about 0.05µg of each substance in 1mL, inject six injections continuously, the S / N is greater than 10.0, and the RSD% is less than 15%. This concentration is used as the quantitative limit of the detection method. The data are shown in Table 7, and the relevant spectra are shown in the attached Figure 3 .

[0044] Table 7 Quantitation limit test data

[0045] Example 4: Determination of detection limit Take appropriate amount of trefarotene reference substance and each impurity reference substance to prepare sample solution containing about 0.025µg of each substance per 1mL, inject three times continuously, and the S / N is greater than 3.0. This concentration is used as the detection limit of this detection method. The data are shown in Table 8.

[0046] Table 8 Detection limit test data

[0047] Example 5: Linearity and range tests Accurately weigh an appropriate amount of trefarotene reference substance, add methanol to dissolve and dilute to prepare a stock solution of a certain concentration; accurately measure an appropriate amount of the stock solution to prepare a series of gradient concentration solutions, which are 0.02%, 0.2%, 1%, 10%, 50%, 100% and 150%, respectively. With the solution concentration as the abscissa and the peak area as the ordinate, perform linear regression and obtain the linear equation. The results are shown in Table 9, and the linear relationship diagram is shown in the attached Figure 4 .

[0048] Table 9 Trifarotene linearity test results

[0049] As shown in Table 9, the linear concentration range of trefarotene is 0.051-386.061 µg / mL.

[0050] Accurately weigh an appropriate amount of each impurity reference substance, add methanol to dissolve and dilute to prepare a certain concentration of stock solution; accurately measure an appropriate amount of stock solution to prepare a series of gradient concentration solutions, which are 0.02%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25% and 0.5% of the sample concentration, respectively. With the solution concentration as the abscissa and the peak area as the ordinate, perform linear regression and obtain the linear equation. The results are shown in Table 10.

[0051] Table 10 Impurity linearity test results

[0052] As shown in Table 10, the linear range of Rtd-002-IMP4 is 0.058~1.455µg / mL; the linear range of Rtd-002-IMP5 is 0.055~1.381µg / mL; the linear range of Rtd-002-IMP3 is 0.051~1.277µg / mL; the linear range of Rtd-002-INT2 is 0.053~1.326µg / mL; 1The linear range is 0.053~1.325µg / mL; the linear range of Rtd-002-IMP2 is 0.053~1.277µg / mL; the linear range of Rtd-002-A is 0.058~1.459µg / mL; the linear range of Rtd-002-B-IMP2 is 0.056~1.389µg / mL; the linear range of Rtd-002-B is 0.054~1.354µg / mL.

[0053] Example 6: System Precision Test The 0.1% solution prepared under the linear test was injected five times continuously and back-injected once at the end of the sequence. The chromatogram was recorded and the precision of the system was examined. The results are shown in Table 11.

[0054] Table 11 System precision test results

[0055] The results showed that the RSD% of the peak areas of each impurity in trefarotene were all less than 5.0%, and the RSD% of the retention time were all less than 2.0%, indicating that the instrument injection precision was good.

[0056] Example 7: Repeatability and accuracy test An impurity reference solution with a concentration of about 100 µg / mL was prepared, and the impurity was added to the trefarotene solution at a concentration of 3*0.02%, 6*0.1% and 3*0.15%, and a total of 12 spiked test solutions were prepared. 5 µL was accurately measured and injected into the liquid chromatograph for recovery determination. The results are shown in Tables 12 to 21.

[0057] Table 12 Repeatability test results

[0058] The results showed that the RSD% of each impurity content in the six spiked test solutions was less than 10.0%, with good repeatability.

[0059] Table 13 Rtd-002-IMP4 accuracy results

[0060] Table 14 Rtd-002-IMP5 accuracy results

[0061] Table 15 Rtd-002-IMP3 accuracy results

[0062] Table 16 Rtd-002-INT2 accuracy results

[0063] Table 17 Rtd-002-IMP1 accuracy results

[0064] Table 18 Rtd-002-IMP2 accuracy results

[0065] Table 19 Rtd-002-A accuracy results

[0066] Table 20 Rtd-002-B-IMP2 accuracy results

[0067] Table 21 Rtd-002-B accuracy results

[0068] The results showed that the recoveries at the 0.02% level were between 70% and 130%, and those at the 0.1% and 0.15% levels were between 80% and 120%; the RSD% of the recoveries at each level were ≤10%, indicating good accuracy.

[0069] Example 8: Solution stability test The stability test was carried out using the reference solution and test solution under the repeatability and accuracy test items. The tests were carried out at 0h, 24h and 48h respectively. The test results are shown in Tables 22 and 23.

[0070] Table 22 Stability results of reference solution

[0071] The results showed that after the reference solution was prepared and placed at room temperature for 24h and 48h, the ratio of the peak area of ​​each impurity to that at 0h was between 90% and 110%, and the reference solution was stable at room temperature within 48h.

[0072] Table 23 Stability results of test solution

[0073] The results showed that after the test solution was prepared and placed at room temperature for 24h and 48h, the ratio of each impurity peak area to 0h was between 90% and 110%, no new impurities were generated, and the test solution was stable within 48h at room temperature.

[0074] Example 9: Durability Test In order to examine the tolerance of this method to changes in conditions, a durability test was conducted. The inspection items included: the proportion of mobile phase, flow rate and column temperature, and the indicators included the theoretical plate number of the main peak, separation degree, and the content of various impurities.

[0075] (1) Change the flow rate: 1.1 mL / min and 1.3 mL / min (2) Changing column temperature: 28°C and 32°C (3) Changing the initial proportion of mobile phase B: 8% B and 12% B The reference solution and sample solution used the reference solution and 0.1% spiked test solution under the repeatability and accuracy test. Accurately pipette 5µL of each solution, inject it into the liquid chromatograph according to the standard conditions and the changed conditions, and record the chromatogram. The results are shown in Table 24.

[0076] Table 24 Durability test results

[0077] The results showed that when the flow rate was between 1.1 and 1.3 mL / min, the column temperature was between 28 and 32 °C, and the initial proportion of mobile phase B was between 8% and 12% B, the main peak retention time, theoretical plate number, and separation degree all changed to a certain extent, and the chromatographic behavior and sample detection results did not change significantly.

[0078] It should be understood by those skilled in the art that the embodiments of the present invention described above and shown in the accompanying drawings are only examples and do not limit the present invention. The advantages of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments, and the embodiments of the present invention may be deformed or modified in any way without departing from the principles.

Claims

1. A method for detecting related substances in trefarotene raw materials, characterized in that: The method for detecting related substances in the trefarotene raw material drug comprises: By means of high performance liquid chromatography, a predetermined concentration of the to-be-detected trefarotene raw material drug is gradient eluted through a mixed mobile phase of mobile phase A and mobile phase B, and a chromatogram of the quantitative limit of trefarotene and each impurity solution is measured; the mobile phase A is a formic acid aqueous solution with a volume ratio of 0.05 to 1; the mobile phase B is a formic acid acetonitrile solution with a volume ratio of 0.05 to 1; the initial ratio of mobile phase A to mobile phase B during the elution process is 88 to 92:12 to 8; The specific gradient elution process is as follows: within 0 to 12 minutes, the ratio of mobile phase A to mobile phase B is gradually changed from the initial ratio to 50 to 60:40 to 50 at a uniform speed; Within 12 to 20 minutes, the ratio of mobile phase A to mobile phase B is gradually changed from 50 to 60:40 to 50 to 40 to 50:50 to 60 at a uniform rate; Within 20 to 30 minutes, the ratio of mobile phase A to mobile phase B gradually changes from 40 to 50:50 to 60 to 1-10:90-99 at a constant speed; Within 30 to 35 minutes, the ratio of mobile phase A to mobile phase B remains constant at 1 to 10:90 to 99; In 35-36 minutes, the ratio of mobile phase A to mobile phase B was gradually changed from 1-10:90-99 to the initial ratio; During 36 to 45 minutes, the ratio of mobile phase A to mobile phase B remained unchanged from the initial ratio; The substance content in trefarotene raw material was obtained by external standard method, and the remaining unknown impurities were obtained by area normalization method.

2. The method for detecting related substances in the trefarotene bulk drug according to claim 1, characterized in that: During the gradient elution process, the initial ratio of mobile phase A to mobile phase B is 85-95:5-15.

3. The method for detecting related substances in the trefarotene bulk drug according to claim 1 or 2, characterized in that: Pipette 1 mL of formic acid into 1000 mL of water, shake well, and degas by ultrasonication to obtain the mobile phase A.

4. The method for detecting related substances in trefarotene bulk drug according to claim 1 or 2, characterized in that: Pipette 1 mL of formic acid into 1000 mL of acetonitrile, shake well, and perform ultrasonic degassing to obtain the mobile phase B.

5. The method for detecting related substances in trefarotene bulk drug according to claim 1 or 2, characterized in that: The HPLC conditions at least meet at least one of the following conditions: The length of the chromatographic column in the high performance liquid chromatography method used is 50 mm to 250 mm; The inner diameter of the chromatographic column is 3 mm to 6 mm; The filler particle size of the chromatographic column is 2 μm to 6 μm; The flow rate of the chromatographic column is 0.5 to 2 mL / min; The column temperature of the chromatographic column is 25-35°C; The detection wavelength of the chromatographic column is 190-400 nm; The injection volume of the chromatographic column is 5 to 10 μL.

6. The method for detecting related substances in trefarotene bulk drug according to claim 5, characterized in that: The HPLC conditions include: flow rate of 1.2 mL / min; column temperature of 30° C.; detection wavelength of 296 nm; and injection volume of 5 μL.

7. The method for detecting related substances in trefarotene bulk drug according to claim 1, characterized in that: The related substances include Rtd-002-IMP1: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(1H-pyrrol-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid; Rtd-002-IMP2: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidin-1-yl)-[1,1':3',1'-triphenyl]-4-carbonitrile; Rtd-002-IMP3: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidin-1-yl)-[1,1':3',1'-triphenyl]-4-carboxamide; Rtd-002-IMP4: 4'-(2-hydroxyethoxy)-[1,1'-biphenyl]-4-carboxylic acid; Rtd-002-IMP5: 4'-(2-hydroxyethoxy)-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid; Rtd-002-INT2: 4'-hydroxy-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid ethyl ester; Rtd-002-A: 4'-(2-acetoxyethoxy)-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid ethyl ester; Rtd-002-B: 4'-(2-acetoxyethoxy)-3'-(tert-butyl)-4'-(pyrrolidin-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid ethyl ester; Rtd-002-B-IMP2: 3''(tert-butyl)-4'-(2-hydroxyethoxy)-4''(pyrrolidin-1-yl)-[1,1':3',1''-triphenyl]-4-carboxylic acid ethyl ester; at least one of the following.

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