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] This application belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting related substances in trofarotine raw material, wherein the detection method uses high performance liquid chromatography to quantitatively analyze nine related substances. Background Technology

[0002] Trifarotene, chemically named 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidine-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid, has the molecular formula C1. 29 H 33 NO4, with a molecular weight of 459.59, has the following chemical structural formula:

[0003] Trafarotin was developed by the French company Galderma and approved by the FDA in the United States in October 2019. This drug belongs to the fourth-generation retinoid class of antibiotics and can selectively activate retinoic acid receptor γ (RAR-γ) in the skin, thereby reducing its impact on other retinoic acid receptor subtypes. This selective binding property helps to significantly reduce skin irritation commonly associated with traditional retinoids, improving patient tolerability and treatment adherence.

[0004] Related substances are starting materials, intermediates, byproducts, and degradation impurities introduced during the synthesis and production of trofarotin. Detection of related substances allows for the control of drug quality and safety. Research on related substances is a key aspect of drug quality research, and their content is a direct indicator of drug purity.

[0005] In the prior art, Chinese patent CN117451892A discloses a method for detecting related substances in low-concentration trefaridine cream. This method has a running time of 70 minutes, which is relatively long, and it does not address potential organic impurities. Additionally, Chinese patent CN118583981A discloses a method for determining the content of genotoxic impurities in trefaridine raw materials, which effectively controls genotoxic impurities in the raw material. However, there are currently no reports on methods for detecting related substances in trefaridine raw materials. Furthermore, there is no method for determining related substances in trefaridine raw materials with low detection limits, low quantitation limits, high sensitivity, high specificity, and high precision and accuracy. In particular, there is no method for quantitatively detecting substances in trefaridine raw materials. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting related substances in trafarotine raw material, employing high-performance liquid chromatography (HPLC) for the simultaneous detection of nine potential related substances in trafarotine raw material. The technical problem to be solved is to provide a method for detecting related substances in trafarotine raw material with low limits of detection and quantitation, high sensitivity, strong specificity, high precision, and high accuracy.

[0007] Another advantage of this invention is that it provides a method for detecting related substances in trefaridine raw material, characterized in that the method for detecting related substances in trefaridine raw material includes:

[0008] High-performance liquid chromatography (HPLC) was used to perform gradient elution of a predetermined concentration of trafarotine active pharmaceutical ingredient using a mixed mobile phase (Mobile Phase A and Mobile Phase B). Chromatograms of trafarotine and its impurities at their limits of quantitation were obtained. Mobile Phase A was an aqueous formic acid solution with a volume ratio of 0.05–1; Mobile Phase B was an acetonitrile formic acid solution with a volume ratio of 0.05–1; the initial ratio of Mobile Phase A to Mobile Phase B during elution was 88–92:12–8.

[0009] The specific gradient elution process is as follows: within 0 to 12 minutes, the ratio of mobile phase A to mobile phase B gradually changes from the initial ratio to 50 to 60: 40 to 50.

[0010] Within 12 to 20 minutes, the ratio of mobile phase A to mobile phase B gradually changes from 50 to 60: 40 to 50 to 40 to 50: 50 to 60 at a constant rate.

[0011] 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 rate.

[0012] Within 30–35 minutes, the ratio of mobile phase A to mobile phase B remains constant at 1–10:90–99.

[0013] Within 35–36 minutes, the ratio of mobile phase A to mobile phase B gradually changes from 1–10:90–99 back to the initial ratio.

[0014] The ratio of mobile phase A to mobile phase B remains unchanged for 36 to 45 minutes;

[0015] The content of substances in trofarotine raw material was obtained by external standard method, and the remaining unknown impurities were obtained by area normalization method.

[0016] According to one embodiment of this application, in the gradient elution process, the initial ratio of mobile phase A to mobile phase B is 85-95:5-15.

[0017] According to one embodiment of this application, 1 mL of formic acid is transferred to 1000 mL of water, shaken well, and degassed by ultrasonication to obtain the mobile phase A.

[0018] According to one embodiment of this application, 1 mL of formic acid is transferred to 1000 mL of acetonitrile, shaken well, and degassed by ultrasonication to obtain the mobile phase B.

[0019] According to an embodiment of this application, the high-performance liquid chromatography conditions satisfy at least one of the following conditions:

[0020] The length of the chromatographic column used in the high-performance liquid chromatography method is 50 mm to 250 mm;

[0021] The inner diameter of the chromatographic column is 3 mm to 6 mm.

[0022] The particle size of the chromatographic column packing material is 2μm to 6μm;

[0023] The flow rate of the chromatographic column is 0.5–2 mL / min;

[0024] The column temperature of the chromatographic column is 25–35°C;

[0025] The detection wavelength of the chromatographic column is 190–400 nm;

[0026] The injection volume of the chromatographic column is 5–10 μL.

[0027] According to one embodiment of this application, the high-performance liquid chromatography (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.

[0028] According to one embodiment of this application, the related substances include Rtd-002-IMP1: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(1H-pyrrolo-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid;

[0029] Rtd-002-IMP2: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidone-1-yl)-[1,1':3',1'-triphenyl]-4-nitrile;

[0030] Rtd-002-IMP3: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidone-1-yl)-[1,1':3',1'-triphenyl]-4-carboxamide;

[0031] Rtd-002-IMP4: 4'-(2-hydroxyethoxy)-[1,1'-biphenyl]-4-carboxylic acid;

[0032] Rtd-002-IMP5: 4'-(2-hydroxyethoxy)-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid;

[0033] Rtd-002-INT2: Ethyl 4'-hydroxy-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid;

[0034] Rtd-002-A: Ethyl 4'-(2-acetoxyethoxy)-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid ester;

[0035] Rtd-002-B: 4'-(2-acetoxyethoxy)-3'-(tert-butyl)-4'-(pyrrolidone-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid ethyl ester;

[0036] Rtd-002-B-IMP2: at least one of 3''(tert-butyl)-4'-(2-hydroxyethoxy)-4''(pyrrolidine-1-yl)-[1,1':3',1''-triphenyl]-4-carboxylic acid ethyl ester. Attached Figure Description

[0037] Figure 1 It is a chromatogram of the solution with good resolution;

[0038] Figure 2 This is the chromatogram of the test sample solution;

[0039] Figure 3 This is a chromatogram of trofarotine and its various impurities at the limit of quantitation.

[0040] Figure 4 This is a linear relationship graph of trofarotine and its various impurities.

[0041] Figure 5 A flowchart of the self-made trofarotine is shown. Detailed Implementation

[0042] The following description is intended to disclose the present invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention.

[0043] In a preferred embodiment, the high-performance liquid chromatography (HPLC) method for detecting related substances in trefaridine active pharmaceutical ingredient provided by the present invention preferably includes the following steps:

[0044] (1) Solution preparation

[0045] Accurately weigh an appropriate amount of trafarotine sample, dissolve it in methanol, and quantitatively dilute it to prepare a solution containing approximately 250 μg of trafarotine per mL, which is used as the test solution. Accurately weigh an appropriate amount of each impurity reference standard, dissolve it in solvent, and quantitatively dilute it to prepare a mixed solution containing approximately 0.25 μg of each impurity reference standard per mL, which is used as the impurity reference standard solution.

[0046] In one example, the trefarotin sample can be any trefarotin selected from the prior art, such as the commercially available French-made Galderma Acne Cream 4th Generation Trefarotin; it can also be synthesized using existing technologies.

[0047] refer to Figure 5 In one example, the synthesis of trofarotine includes:

[0048] (S1.1) Add 20 g (1 eq, 134 mmol) of 2-tert-butylaniline to a 500 ml three-necked flask, add 200 ml (10 ml / g) of THF and stir thoroughly to dissolve. Cool the system to 0 °C. Add 64.69 g (1 eq, 134 mmol) of tetrabutylammonium tribromide in five batches, 12.938 g (0.2 eq) per batch, with each batch added over 5 min and 10 min intervals between batches. After the addition is complete, slowly raise the temperature to room temperature and stir for 10 min. Slowly add water dropwise to quench the reaction. Wash with ethyl acetate, let stand, and wait... The liquid completely separated into layers. The EA phase was collected, and EA was added back to the aqueous phase. The mixture was stirred and washed thoroughly. After standing, the liquid completely separated into layers. The EA phase was collected again, and EA was added back to the aqueous phase. The mixture was stirred and washed thoroughly. After standing, the liquid completely separated into layers. The organic phase was collected, and the mixture was washed twice with sodium sulfite aqueous solution, once with sodium bicarbonate aqueous solution, twice with water, and once with sodium chloride aqueous solution. After drying with anhydrous sodium sulfate, the mixture was distilled under reduced pressure to obtain 31.94 g of crude product, which was a brown oily product. This product was not purified to obtain intermediate Rtd-002-int1-A. 1 H NMR (400 MHz, Chloroform-d) δ 7.32 (d, J = 2.3 Hz, 1H), 7.13 (dd, J = 8.4, 2.3 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 3.83 (s, 2H), 1.41 (s, 9H). LC-MS m / z(M+ACN) + =268.9, tR=2.679.

[0049] (S1.2) 1 g (1 eq, 4.4 mmol) of intermediate Rtd-002-int1-A was added to a 100 mL single-necked flask. 10 mL of dioxane (10 mL / g) was added and stirred thoroughly to dissolve. 1.34 g (1.2 eq, 5.2 mmol) of pinacol diboronate, 1.29 g (3 eq, 13 mmol) of potassium acetate, and 160 mg (0.05 eq, 0.2 mmol) of dppf palladium dichloride were added. The reaction system was protected with nitrogen and heated to 90 °C with stirring for 16 h. The system was cooled, and the reaction was quenched with water. Ethyl acetate was added for extraction, and the organic phase was collected. The organic phase was then extracted with water, followed by extraction with saturated sodium chloride aqueous solution. The mixture was dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The product was purified by MPLC (PE:EA = 15%) to obtain 690 mg of the product, thus yielding intermediate Rtd-002-C. 1 H NMR (400 MHz, DMSO-d6) δ 7.42 (d, J = 1.5 Hz, 1H), 7.23 (dd, J = 8.0, 1.4 Hz, 1H), 6.61 (d,J = 7.9 Hz, 1H), 5.21 (s, 2H), 1.26 (s, 12H), 1.18 (s, 9H). LC-MS m / z (M+H) + =276.0,t R =2.769.

[0050] (S2.1) Add 20g (1 eq, 102 mmol) of cyanobiphenol to a 500ml three-necked flask, add 200ml of glacial acetic acid and stir thoroughly. Add 5.02g (0.5 eq) of concentrated sulfuric acid to the system and stir thoroughly. Maintain the temperature of the system at 18-20℃. Add 23.07g (1 eq, 102 mmol) of N-iodosuccinimide to the above reaction system in ten batches, each batch being 2.307g (0.1 eq). During the addition process, maintain the temperature at 18-20℃, with a 30min interval between each batch. After the addition is complete, continue stirring for 5h. Slowly pour the reaction system into water and stir for 1h. Filter, rinse the filter cake with water, collect the filter cake, and dry it in a vacuum drying oven to obtain 32g of Rtd-002-int2-A, which is an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 8.08 (d, J= 2.3 Hz, 1H), 7.91 – 7.79 (m, 4H), 7.65 (dd, J = 8.5, 2.3 Hz, 1H), 7.01 (d,J = 8.5 Hz, 1H). LC-MS m / z (MH)-=319.9,tR =2.424.

[0051] (S2.2) Add 32g (1 eq, 100 mmol) of intermediate Rtd-002-int2-A to a 1L single-necked flask, add 160ml of ethanol and stir thoroughly. Separately, take 20g (5 eq, 500 mmol) of sodium hydroxide, add 160ml of water and stir thoroughly to dissolve. After cooling to room temperature, slowly add it to the above system. The system is protected by nitrogen gas. The reaction system is heated to 100℃ and stirred for 16h. After the reaction was complete, the temperature was slowly lowered to room temperature, and the system was distilled under reduced pressure to remove ethanol. 60 ml of 6 mol / L hydrochloric acid was prepared and added dropwise to the system. The pH was checked with pH paper; if the pH was less than 5, a large amount of solid would precipitate. The mixture was filtered, the filter cake was rinsed with water, and the cake was collected. 200 ml of water was added to the filter cake, and the mixture was heated to 50°C and stirred thoroughly for 2 hours. The temperature was slowly lowered to 25°C, and the mixture was filtered again. The filter cake was rinsed with water, collected, and dried in a vacuum drying oven to obtain 32 g of crude Rtd-002-int2-B, a white solid. 96 ml of acetone (3 ml / g) was added, and the mixture was heated to 70°C and stirred thoroughly to dissolve. Hot filtration was performed to remove insoluble impurities. The filtrate was slowly lowered to -20°C, and a large amount of solid precipitated. The mixture was filtered, and the filter cake was not rinsed with the mother liquor or with clean acetone. The solid was collected to obtain 25 g of the intermediate Rtd-002-int2-B, a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 10.63 (s,1H), 8.05 (d, J = 2.3 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.2 Hz,2H), 7.62 (dd, J = 8.5, 2.3 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H). LC-MS m / z (MH) - =338.9,t R =2.063.

[0052] (S2.3) 25 g (1 eq, 100 mmol) of intermediate Rtd-002-int2-B was added to a 1 L single-necked flask, and 250 ml of ethanol was added and stirred thoroughly to dissolve it. 25 ml of concentrated sulfuric acid was added to the system, and the system was protected with nitrogen. The system was heated to 100 °C and stirred for 16 h. The reaction system was slowly cooled to room temperature, and 250 ml of water was slowly added dropwise to the system and stirred for 30 min. A solid precipitated out. The solid was filtered, the filter cake was washed with water, and the solid was collected and dried in a vacuum drying oven to obtain 22 g of crude product, which was an off-white solid. 88 ml of toluene (3 ml / g) was added, and the mixture was heated to 110 °C and stirred thoroughly to dissolve it. The temperature was slowly lowered to -20 °C, and a large amount of solid precipitated out. The solid was filtered, and the filter cake was collected to obtain 20 g of intermediate Rtd-002-int2, which was an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.65 (s,1H), 8.06 (d, J = 2.3 Hz, 1H), 8.02 – 7.98 (m, 2H), 7.80 – 7.74 (m, 2H), 7.63(dd, J = 8.4, 2.3 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H). LC-MS m / z (MH) - =366.9,t R =2.720.

[0053] (S3.1) Add 20g (1 eq, 54 mmol) of intermediate Rtd-002-int2 to a 500ml single-necked flask, add 200ml of DMF and stir thoroughly to dissolve, add 24g (3.2 eq, 174 mmol) of potassium carbonate to the system, under nitrogen protection, stir thoroughly for 30min, add 11.47g (1.3 eq, 69 mmol) of 2-bromoethyl acetate to the system, change to nitrogen protection, heat to 60℃ and stir for 3h. After the reaction was complete, the temperature was slowly lowered to room temperature. The reaction system was slowly poured into 1 L of water and stirred thoroughly for 10 min. The mixture was filtered, the filter cake was rinsed with water, and the filter cake was collected. 500 ml of ethyl acetate was added to redissolve the filter cake. 500 ml of water was added to the organic phase and stirred thoroughly. The mixture was allowed to stand and separate into layers, and the upper organic phase was collected. 500 ml of water was added to the organic phase and stirred thoroughly. The mixture was allowed to stand and separate into layers, and the upper organic phase was collected. 200 ml of saturated sodium chloride aqueous solution was added to the organic phase and stirred thoroughly. The mixture was allowed to stand and separate into layers, and the organic phase was collected. Anhydrous sodium sulfate was added to the organic phase and dried. The solvent was removed by vacuum distillation to obtain 22.2 g of crude intermediate Rtd-002-A, which was an off-white solid. 22.2 g of solid was added to 66 ml of ethanol (3 ml / g), heated to 85 °C and stirred to dissolve. The mixture was then slowly cooled to -25 °C, resulting in the precipitation of a large amount of solid. The solid was filtered, and the filter cake was washed with the mother liquor and clean ethanol. The filter cake was collected to obtain 21.5 g of off-white solid, which was intermediate Rtd-002-A. 1 HNMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 2.3 Hz, 1H), 8.03 – 7.97 (m, 2H), 7.82– 7.78 (m, 2H), 7.76 (dd, J = 8.6, 2.4 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 4.41 – 4.29 (m, 6H), 2.06 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H).LC-MS m / z (M+ACN) + =496.0,t R =3.051.

[0054] (S3.2) Add 3.16 g (3 eq, 11.9 mmol) of potassium phosphate trihydrate to a 100 ml three-necked flask, add 3 ml of water and stir thoroughly to dissolve. Add 1.8 g (1 eq, 3.9 mmol) of Rtd-002-A and 1.3 g (1.2 eq, 4.7 mmol) of Rtd-002-C, add 15 ml of DMF and stir thoroughly to dissolve. Purge the system under nitrogen protection. Add 97 mg (0.03 eq, 0.1 mmol) of [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex. Purge the system under nitrogen protection and stir at room temperature for 5 h. Slowly pour the reaction solution into water, wash with EA, collect the organic phase, wash with water, wash with saturated brine, dry with anhydrous sodium sulfate, remove the organic solvent by vacuum distillation, and purify by MPLC (PE:EA = 20%) to obtain 1.5 g of white solid, which is Rtd-002-D. 1 H NMR(400 MHz, DMSO-d6) δ 8.02 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 8.3 Hz, 2H), 7.60(s, 2H), 7.40 (d, J = 2.0 Hz, 1H), 7.22 – 7.16 (m, 2H), LC-MS m / z(M+H) + =475.3,t R =3.179.

[0055] (S3.3) 1.5 g (1 eq, 3.1 mmol) of Rtd-002-D was added to a 100 ml single-necked flask, 15 ml of NMP was added and stirred thoroughly to dissolve, 243 mg (3.2 eq, 10.1 mmol) of sodium hydride and 2.39 g (3.5 eq, 11 mmol) of 1,4-dibromobutane were added, and the mixture was heated to 100 °C and stirred for 16 h. The reaction solution was slowly poured into water to quench the reaction, and EA was added as an eluent. The EA phase was collected, and the EA phase was further eluented with water and saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The mixture was purified by MPLC (PE:EA = 20%) to obtain 524 mg of white solid, which was Rtd-002-B. 1H NMR (400 MHz, DMSO-d6) δ 8.03 – 7.97 (m, 2H),7.88 – 7.83 (m, 2H), 7.70 (dd, J = 8.5, 2.5 Hz, 1H), 7.67 (d, J = 2.4 Hz,1H), 7.57 (d, J = 1.9 Hz, 1H), 7.48 – 7.41 (m, 2H), 7.25 (d, J = 8.6 Hz, 1H), 4.37 – 4.26 (m, 6H), 2.96 (s, 4H), 1.98 (s, 3H), 1.91 (d, J = 6.2 Hz, 4H),1.42 (s, 9H), 1.34 (t, J = 7.1 Hz, 3H). LC-MS m / z (M+H) + =530.3,t R =6.369.

[0056] (S3.4) Add 500 mg (1 eq, 0.9 mmol) of Rtd-002-B to a 100 mL single-necked flask, add 5 mL of ethanol and stir thoroughly. Separately, dissolve 113 mg (3 eq, 2.7 mmol) of sodium hydroxide in 5 mL of water and stir thoroughly. After cooling to room temperature, add the solution to the above system. Purge the system with nitrogen for protection and stir at 70 °C for 3 h. At 70 °C, slowly add 3 mL of 1 mol / L hydrochloric acid aqueous solution to the reaction system. Check the pH; if it is between 5.5 and 6.0, a solid will precipitate. Filter while hot, rinse the filter cake with water, collect the filter cake, and dry it in a vacuum drying oven to obtain 347 mg of trefaroline, which is an off-white solid.

[0057] 1 H NMR (400 MHz, DMSO-d6) δ 7.99 (d, J = 8.4 Hz, 2H), 7.84 – 7.78 (m,2H), 7.68 (dd, J = 8.5, 2.5 Hz, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.59 (d, J =1.9 Hz, 1H), 7.51 – 7.42 (m, 2H), 7.23 (d, J = 8.6 Hz, 1H), 4.82 (s, 1H), 4.10 (t, J = 5.2 Hz, 2H), 3.73 (d, J = 5.2 Hz, 2H), 2.96 (s, 4H), 1.89 (d, J= 3.5 Hz, 4H). LC-MS m / z (M+H) + =460.6,tR =2.547.

[0058] (2) Inject the test solution and the impurity reference solution into the liquid chromatograph separately, record the chromatograms, and calculate the peak area using the external standard method. Preferably, the chromatographic conditions are as follows:

[0059] The chromatographic column was a Waters XBridge BEH C18 4.6*150mm, 3.5μm.

[0060] The detection wavelength is 190–400 nm, preferably 296 nm.

[0061] The flow rate is 1.1–1.3 mL / min, preferably 1.2 mL / min.

[0062] The column temperature is 25–35℃, preferably 30℃.

[0063] The injection volume is 5–10 μL, preferably 5 μL.

[0064] Mobile phase A is a 0.1% (v / v) aqueous solution of formic acid, which can be obtained by transferring 1 mL of formic acid to 1000 mL of water, shaking well, and then degassing by sonication.

[0065] Mobile phase B is a 0.1% formic acid acetonitrile solution (v / v), which can be obtained by transferring 1 mL of formic acid to 1000 mL of acetonitrile, shaking well, and then degassing by sonication.

[0066] Perform gradient elution according to Table 1:

[0067] Table 1

[0068] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 12 55 45 20 45 55 30 5 95 35 5 95 36 90 10 45 90 10

[0069] It is worth mentioning that the method for detecting related substances in trafarotine API provided by this invention can simultaneously detect nine potential related substances in trafarotine API during high-performance liquid chromatography (HPLC) analysis. The separation between impurities and between impurities and the main component is good, and the response of each component is high. It has low limits of detection and quantitation, high sensitivity, strong specificity, and high precision and accuracy. This method can rapidly and easily detect and accurately quantify related substances in trafarotine API, greatly improving the efficiency and accuracy of drug analysis and providing strong technical support for the quality control of trafarotine API.

[0070] As an example, when selecting drugs and reagents, the following are selected: trafarotin (Rtd-002) reference standard with a content of 98.99%; Rtd-002-IMP1 reference standard with a purity of 81.87%; Rtd-002-IMP2 reference standard, preferably with a purity of 97.76%; Rtd-002-IMP3 reference standard, preferably with a purity of 98.71%; Rtd-002-IMP4 reference standard, preferably with a purity of 99.46%; Rtd-002-IMP5 reference standard, preferably with a purity of 94.81%; Rtd-002-INT2 reference standard, preferably with a purity of 96.92%; Rtd-002- Reference standard A, preferably with a purity of 94.51%; Rtd-002-B reference standard, preferably with a purity of 96.94%; Rtd-002-B-IMP2 reference standard, preferably with a purity of 92.61%; sodium chloride, preferably analytical grade, 3A Chemical; hydrochloric acid, preferably analytical grade, Sinopharm Chemical Reagent Co., Ltd.; sodium hydroxide, preferably analytical grade, Shanghai Boer Chemical Reagent Co., Ltd.; formic acid, preferably chromatographic grade, DAMA-Beta; methanol, preferably chromatographic grade, DAMA-Beta; acetonitrile, preferably chromatographic grade, DAMA-Beta; ultrapure water, preferably Watson's. Furthermore, when selecting instruments, the preferred options are: Agilent 1260 high-performance liquid chromatograph, Shimadzu AUW220D electronic balance, and electric thermostatic drying oven.

[0071] More preferably, when selecting liquid chromatography conditions, a Waters XBridge BEH C18 column (4.6*150mm, 3.5μm) is used. A mobile phase A is 0.1% formic acid aqueous solution (v / v), and a mobile phase B is 0.1% formic acid acetonitrile solution (v / v). Gradient elution is performed according to Table 2. The detection wavelength is 296nm, the flow rate is 1.2mL / min, and the column temperature is 30℃. 5μL of the test solution and the impurity reference solution are accurately measured and injected into the liquid chromatograph, respectively, and the chromatograms are recorded.

[0072] Table 2

[0073] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 12 55 45 20 45 55 30 5 95 35 5 95 36 90 10 45 90 10

[0074] When detecting related substances in trefaridine, first accurately weigh approximately 25 mg of trefaridine, place it in a 100 mL volumetric flask, dissolve it in methanol, dilute to the mark, and shake well to prepare the test solution. Accurately weigh appropriate amounts of each impurity reference standard, dissolve them in solvent, and quantitatively dilute to prepare a mixed solution containing approximately 0.25 μg of each impurity reference standard per mL, which is used as the impurity reference standard solution. Accurately inject 5 μL each of the test solution and impurity reference standard solution into the liquid chromatograph and record the chromatograms. Use the external standard method to quantify known impurities and the area normalization method to determine unknown impurities. The detection results are shown in Table 3 below:

[0075] Table 3

[0076]

[0077] I. Verification was performed through the following examples.

[0078] Example 1: Specificity

[0079] (1) Blank solvent: methanol;

[0080] (2) Test solution: Weigh about 25 mg of trefaridine sample accurately, place it in a 100 mL volumetric flask, add methanol to dissolve and dilute to the mark, and shake well.

[0081] (3) Separation solution: Weigh appropriate amounts of trefaridine reference standard and each impurity reference standard, dissolve them in solvent and dilute quantitatively to prepare a mixed solution containing approximately 0.25 μg of each impurity and 250 μg of trefaridine per 1 mL, and shake well.

[0082] (4) Single impurity positioning solution: Take an appropriate amount of each impurity reference standard, dissolve and dilute it with solvent to a certain concentration, and shake well.

[0083] Accurately measure 5 μL of each of the above solutions and inject them into the liquid chromatograph, then record the chromatograms. The results are shown in Table 4, and relevant chromatograms are attached. Figure 1 and Figure 2 .

[0084] Table 4. Results of Specificity Experiment

[0085]

[0086] The results showed that under these chromatographic conditions, the baseline was stable, and the blank solvent did not interfere with the determination of this product. In the resolution solution, the peak shapes of each substance were good, with the theoretical plate number of trefaridine's main peak being 120426.57. The resolution between the main peak and adjacent peaks was 1.63, ≥1.5, indicating good separation among the impurities. The retention time ratios of each impurity in the resolution solution and the positioning solution were all between 0.98 and 1.02, indicating good specificity.

[0087] Example 2: Destructive Test

[0088] To investigate whether the potential degradation products of trefaridine could be detected under the selected chromatographic conditions, the product was subjected to harsh conditions such as high temperature, high humidity, acid, alkali, and oxidation. The destroyed samples were dissolved in methanol to prepare test solutions. 5 μL of each of these test solutions was accurately measured and injected into the liquid chromatograph, and the chromatograms were recorded. Specific methods are shown in Tables 5 and 6.

[0089] Table 5 Results of the destructive test

[0090]

[0091] Mass balance * = (content of main component after destruction + content of impurities after destruction) / (content of main component before destruction + total impurities before destruction).

[0092] Table 6. Impurities in the test sample solution under various destructive conditions.

[0093]

[0094] Total impurities* = the sum of all single impurities with a content ≥ 0.01%.

[0095] The results showed that the product degraded to varying degrees under acid, alkali, and oxidative conditions. Oxidative degradation had the greatest impact, followed by alkali degradation. The product was relatively stable under high temperature, high temperature, and acidic conditions. Degradation products generated under all degradation conditions were detectable. The resolution between the main component and adjacent impurities was greater than 1.5, and the resolution between all impurities was greater than 1.0. The mass balance was within the range of 0.95–1.05, and the purity of the main peak was above 0.990, indicating material conservation.

[0096] Example 3: Determination of the limit of quantitation

[0097] Take appropriate amounts of trefaridine reference standard and each impurity reference standard, and prepare sample solutions containing approximately 0.05 µg of each substance per 1 mL. Inject six consecutively. The S / N ratio should be greater than 10.0 and the RSD% less than 15%. This concentration is used as the limit of quantitation for this detection method. Data are shown in Table 7, and relevant spectra are attached. Figure 3 .

[0098] Table 7. Limit of Quantitation Test Data

[0099]

[0100] Example 4: Determination of the detection limit

[0101] Take appropriate amounts of trefaridine reference standard and each impurity reference standard, and prepare sample solutions containing approximately 0.025 µg of each substance per 1 mL. Inject the solution three times consecutively. The S / N ratio should be greater than 3.0 for each solution. This concentration is taken as the detection limit of this method. The data are shown in Table 8.

[0102] Table 8 Detection Limit Test Data

[0103]

[0104] Example 5: Linearity and Range Test

[0105] Accurately weigh an appropriate amount of trefaridine reference standard, dissolve and dilute it in methanol to prepare a stock solution of a certain concentration; accurately measure an appropriate amount of the stock solution to prepare a series of solutions with gradient concentrations of 0.02%, 0.2%, 1%, 10%, 50%, 100%, and 150%. Perform linear regression with solution concentration as the x-axis and peak area as the y-axis, and derive the linear equation. The results are shown in Table 9, and the linear relationship graph is attached. Figure 4 .

[0106] Table 9 Results of linearity test of trefaridine

[0107]

[0108] As shown in Table 9, the linear concentration range of trofarotine is 0.051–386.061 µg / mL.

[0109] Accurately weigh appropriate amounts of each impurity reference standard, dissolve and dilute with methanol to prepare stock solutions of a certain concentration; accurately measure appropriate amounts of the stock solutions to prepare a series of solutions with gradient concentrations of 0.02%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, and 0.5% of the sample concentration. Perform linear regression with solution concentration as the x-axis and peak area as the y-axis, and derive the linear equation. The results are shown in Table 10.

[0110] Table 10 Results of linearity tests for each impurity

[0111]

[0112] Table 10 shows that the linear range of Rtd-002-IMP4 is 0.058–1.455 µg / mL; the linear range of Rtd-002-IMP5 is 0.055–1.381 µg / mL; the linear range of Rtd-002-IMP3 is 0.051–1.277 µg / mL; the linear range of Rtd-002-INT2 is 0.053–1.326 µg / mL; and the linear range of Rtd-002-IMP... The linear range of Rtd-002-IMP2 is 0.053–1.325 µg / mL; the linear range of Rtd-002-A is 0.053–1.277 µg / mL; the linear range of Rtd-002-A is 0.058–1.459 µg / mL; the linear range of Rtd-002-B-IMP2 is 0.056–1.389 µg / mL; and the linear range of Rtd-002-B is 0.054–1.354 µg / mL.

[0113] Example 6: System Precision Test

[0114] The 0.1% solution prepared according to the linearity test was injected five times consecutively, with a final injection at the end of the sequence. The chromatograms were recorded to examine the system precision. The results are shown in Table 11.

[0115] Table 11 System precision test results

[0116]

[0117] The results showed that the RSD% of the peak area of ​​each impurity in trofarotine was less than 5.0%, and the RSD% of the retention time was less than 2.0%, indicating good instrument injection precision.

[0118] Example 7: Repeatability and Accuracy Test

[0119] A reference solution of impurities with a concentration of approximately 100 µg / mL was prepared. The impurities were added to the trefaridine solution at concentrations of 3*0.02%, 6*0.1%, and 3*0.15%, resulting in 12 spiked test solutions. 5 µL of each solution was accurately injected into the liquid chromatograph for recovery determination. The results are shown in Tables 12 to 21.

[0120] Table 12 Repeatability Test Results

[0121]

[0122] The results showed that the RSD% of each impurity in the six spiked test solutions was less than 10.0%, indicating good repeatability.

[0123] Table 13 Accuracy Results of Rtd-002-IMP4

[0124]

[0125] Table 14 Accuracy Results of Rtd-002-IMP5

[0126]

[0127] Table 15 Accuracy Results of Rtd-002-IMP3

[0128]

[0129] Table 16 Accuracy Results of Rtd-002-INT2

[0130]

[0131] Table 17 Accuracy Results of Rtd-002-IMP1

[0132]

[0133] Table 18 Accuracy Results of Rtd-002-IMP2

[0134]

[0135] Table 19 Accuracy Results of Rtd-002-A

[0136]

[0137] Table 20 Accuracy Results of Rtd-002-B-IMP2

[0138]

[0139] Table 21 Accuracy Results of Rtd-002-B

[0140]

[0141] The results showed that the recoveries at the 0.02% level were between 70% and 130%, and the recoveries at the 0.1% and 0.15% levels were between 80% and 120%. The RSD% of the recoveries at each level was ≤10%, indicating good accuracy.

[0142] Example 8: Solution Stability Test

[0143] Stability was investigated using the reference solution and the test solution under the repeatability and accuracy test. The tests were conducted at 0h, 24h and 48h, respectively. The test results are shown in Tables 22 and 23.

[0144] Table 22 Stability results of reference solutions

[0145]

[0146] The results showed that after the reference solution was prepared and left at room temperature for 24 h and 48 h, the ratio of the peak area of ​​each impurity to that at 0 h was between 90% and 110%, and the reference solution was stable at room temperature for 48 h.

[0147] Table 23 Stability results of the test solution

[0148]

[0149] The results showed that after the test solution was prepared and left at room temperature for 24 h and 48 h, the ratio of the peak area of ​​each impurity to that at 0 h was between 90% and 110%, and no new impurities were generated. The test solution was stable at room temperature for 48 h.

[0150] Example 9: Durability Test

[0151] To assess the method's tolerance to changing conditions, a robustness test was conducted. The test parameters included the mobile phase ratio, flow rate, and column temperature, with indicators including the theoretical plate number of the main peak, resolution, and the content of each impurity.

[0152] (1) Change the flow rate: 1.1 mL / min and 1.3 mL / min

[0153] (2) Change the column temperature: 28℃ and 32℃

[0154] (3) Change the initial proportion of mobile phase B: 8%B and 12%B

[0155] The reference solution and sample solution used were the reference solution and 0.1% spiked test solution from the repeatability and accuracy test. 5 µL of each solution was precisely transferred and injected into the liquid chromatograph under both standard and modified conditions, and the chromatograms were recorded. The results are shown in Table 24.

[0156] Table 24 Durability Test Results

[0157]

[0158] The results showed that when the flow rate varied from 1.1 to 1.3 mL / min, the column temperature from 28 to 32 °C, and the initial proportion of mobile phase B varied from 8% to 12%B, the retention time of the main peak, the theoretical plate number, and the resolution all changed to some extent, but the chromatographic behavior and sample detection results did not change significantly.

[0159] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the accompanying drawings are merely examples and do not limit the invention. The advantages of the present invention have been fully and effectively realized. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments; any variations or modifications can be made to the implementation of the present invention without departing from these principles.

Claims

1. A method for detecting related substances in trofarotin raw material, characterized in that, The method for detecting related substances in the trofarotine active pharmaceutical ingredient includes: High-performance liquid chromatography (HPLC) was used to perform gradient elution of a predetermined concentration of trafarotine active pharmaceutical ingredient using a mixed mobile phase (Mobile Phase A and Mobile Phase B). Chromatograms of trafarotine and its impurities at their limits of quantitation were obtained. Mobile Phase A was an aqueous formic acid solution with a volume ratio of 0.05–1; Mobile Phase B was an acetonitrile formic acid solution with a volume ratio of 0.05–1; the initial ratio of Mobile Phase A to Mobile Phase B during elution was 88–92:12–8. The specific gradient elution process is as follows: within 0 to 12 minutes, the ratio of mobile phase A to mobile phase B gradually changes from the initial ratio to 50 to 60: 40 to 50. Within 12 to 20 minutes, the ratio of mobile phase A to mobile phase B gradually changes from 50 to 60: 40 to 50 to 40 to 50: 50 to 60 at a constant 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 rate. The ratio of mobile phase A to mobile phase B remains constant at 1–10:90–99 for 30–35 minutes. Within 35–36 minutes, the ratio of mobile phase A to mobile phase B gradually changes from 1–10:90–99 back to the initial ratio. The ratio of mobile phase A to mobile phase B remains unchanged for 36 to 45 minutes; The content of substances in trofarotine 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 trefaridine raw material 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 trefaridine raw material according to claim 1 or 2, characterized in that, Transfer 1 mL of formic acid to 1000 mL of water, shake well, and degas by sonication to obtain the mobile phase A.

4. The method for detecting related substances in trefaridine raw material according to claim 1 or 2, characterized in that, Transfer 1 mL of formic acid to 1000 mL of acetonitrile, shake well, and degas by sonication to obtain the mobile phase B.

5. The method for detecting related substances in trefaridine raw material according to claim 1 or 2, characterized in that, The high-performance liquid chromatography conditions must satisfy at least one of the following conditions: The length of the chromatographic column used in the high-performance liquid chromatography method is 50 mm to 250 mm; The inner diameter of the chromatographic column is 3 mm to 6 mm. The particle size of the chromatographic column packing material is 2μm to 6μm; The flow rate of the chromatographic column is 0.5–2 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 5–10 μL.

6. The method for detecting related substances in trefaridine raw material according to claim 5, characterized in that, The high-performance liquid chromatography (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.

7. The method for detecting related substances in trefaridine raw material according to claim 1, characterized in that, The related substances include Rtd-002-IMP1: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(1H-pyrrolo-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid; Rtd-002-IMP2: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidone-1-yl)-[1,1':3',1'-triphenyl]-4-nitrile; Rtd-002-IMP3: 3'-(tert-butyl)-4'-(2-hydroxyethoxy)-4'-(pyrrolidone-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: Ethyl 4'-hydroxy-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid; Rtd-002-A: Ethyl 4'-(2-acetoxyethoxy)-3'-iodo-[1,1'-biphenyl]-4-carboxylic acid ester; Rtd-002-B: 4'-(2-acetoxyethoxy)-3'-(tert-butyl)-4'-(pyrrolidone-1-yl)-[1,1':3',1'-triphenyl]-4-carboxylic acid ethyl ester; Rtd-002-B-IMP2: at least one of 3''(tert-butyl)-4'-(2-hydroxyethoxy)-4''(pyrrolidine-1-yl)-[1,1':3',1''-triphenyl]-4-carboxylic acid ethyl ester.

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