Method for detecting tretinoin and / or related substances thereof in tretinoin preparation

Abstract

The invention provides a method for detecting tretinoin and / or related substances thereof in a tretinoin preparation, and relates to the technical field of medicine detection. The method comprises the following steps: pre-treating a tretinoin preparation to be detected: mixing the preparation to be detected, a gel breaker and a diluent, carrying out solid-liquid separation operation, and collecting a liquid component which is a sample liquid to be detected; by adopting the pretreatment method, matrix interference in the preparation to be detected can be effectively removed. The to-be-detected sample solution is detected by high performance liquid chromatography, and rapid qualitative and quantitative analysis of tretinoin and seven related substances in the tretinoin preparation can be realized. Wherein the tretinoin and the seven related substances have good peak shapes, the separation degree of each substance is high, the accuracy of a detection result is high, the detection limit is low, the quantitation limit is low, the detection sensitivity is high, and the stability is strong. According to the detection method provided by the invention, the defect of detection of tretinoin and related substances in the tretinoin preparation in the prior art is overcome, and a technical support is provided for inspection and quality control of the tretinoin preparation.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, specifically to a method for detecting retinoic acid and / or related substances in retinoic acid preparations. Background Technology

[0002] Retinoin is an important vitamin A derivative widely used in the treatment of acne, skin aging, and acute promyelocytic leukemia (APL). As a dermatological treatment, tretinoin was first approved for marketing in the United States by the Food and Drug Administration (FDA) in 1971, initially as a topical ointment. Subsequently, various dosage forms, including creams, gels, lotions, tablets, and capsules, were developed, further expanding the clinical application of this drug.

[0003] The chemical structure of retinoic acid contains a polyene side chain with multiple double bonds. These double bonds can exist in cis or trans forms, resulting in various isomers, including but not limited to isotretinoin, 9,13-di-cis-retinoic acid, 11,13-di-cis-retinoic acid, and 9-cis-retinoic acid. These isomers can undergo reversible interconversion under the influence of light or heat. Furthermore, light, heat, and oxidation can also degrade retinoic acid, producing other impurities. These isomeric impurities and degradation impurities seriously affect the safety and efficacy of the drug. For example, the main adverse reactions of isotretinoin include: most common (>10%): dry cheilitis (100%), dry skin (94.97%), facial erythema (66.21%), epistaxis (47.26%), myalgia (38.78%); psychiatric symptoms (25.16%): mood changes, fatigue, insomnia, etc., but mostly short-term; eye problems (8.96%): dry eye, blurred vision (rare); abnormal test results: elevated blood lipids (3.11%), elevated liver enzymes (2.09%), which resolve after discontinuation of the drug. 9,13-cis-retinoic acid may promote liver fibrosis. The most common side effects of 9-cis-retinoic acid include headache and adverse changes in the skin and mucous membranes. Abnormalities frequently reported in clinical chemistry tests include hypercalcemia and lipid abnormalities. Therefore, accurate separation and analysis of retinoic acid impurities are of great significance for drug quality control. Currently, the pharmacopoeias of major countries around the world do not have complete control over the impurity limits of the above-mentioned retinoic acid preparations. For example, the Chinese Pharmacopoeia and the European Pharmacopoeia only control impurity A (isotretinoin) of retinoic acid; the United States Pharmacopeia does not control specific impurities of retinoic acid.

[0004] The analytical methods for retinoic acid listed in pharmacopoeias of various countries are sufficient for the separation and detection of impurity A, but their ability to separate and detect other isomers and degradation impurities is poor. Furthermore, because retinoic acid is primarily used to treat dermatological diseases, common dosage forms are mostly semi-solid preparations such as creams, gels, and lotions. Semi-solid preparations have complex compositions, and retinoic acid often constitutes a low percentage (≤0.1%). Therefore, conventional detection methods often involve sample pretreatment processes such as liquid-liquid extraction, which are cumbersome and time-consuming. Therefore, there is an urgent need for an analytical method that is simple to operate and has excellent impurity separation and detection capabilities. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for detecting retinoic acid and / or its related substances in retinoic acid preparations. The detection method provided by this invention is simple to operate, has high separation degree of retinoic acid and its related substances, and high detection sensitivity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] This invention provides a method for detecting retinoic acid and / or related substances in retinoic acid preparations, comprising the following steps:

[0008] The retinoic acid preparation to be tested, the gelling agent, and the diluent are mixed and extracted, followed by solid-liquid separation. The liquid component is the sample solution to be tested. The diluent includes water and an organic phase. The organic phase includes isopropanol and other organic solvents. The other organic solvents include at least one of methyl tert-butyl ether, ethyl acetate, and tetrahydrofuran.

[0009] The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of retinoic acid and / or related substances;

[0010] The conditions for the high-performance liquid chromatography (HPLC) detection include: a Waters XSelect CSH Fluoro-Phenyl column and a Welch column. PAH column or Waters XSelect HSS PFP column; mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is 0.1–0.5 v / v% formic acid aqueous solution or 0.5–2 v / v% acetic acid aqueous solution, and mobile phase B is methanol, 1–10 v / v% methyl tert-butyl ether-methanol solution or 0.3–3 v / v% methyl tert-butyl ether-isopropanol solution; elution mode is isocratic elution or gradient elution; during isocratic elution, the volume ratio of mobile phase A to mobile phase B is 10:90–90. :10; The gradient elution program is as follows: 0–21 min, the volume fraction of mobile phase A is 80%; 21–30 min, the volume fraction of mobile phase A linearly decreases from 80% to 60%; 30–50 min, the volume fraction of mobile phase A linearly decreases from 60% to 30%; 50–55 min, the volume fraction of mobile phase A linearly increases from 30% to 80%; 55–70 min, the volume fraction of mobile phase A is 80%.

[0011] The related substances include one or more of isotretinoin, 9,13-cis-retinoic acid, 11,13-cis-retinoic acid, avitamin A, 9-cis-retinoic acid, all-trans-4-methoxyretinoic acid, and all-trans-5,6-epoxyretinoic acid.

[0012] Preferably, during the isocratic elution process, the volume ratio of mobile phase A to mobile phase B is 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, or 90:10.

[0013] Preferably, the chromatographic column has a size of 4.6 × 150 mm and a packing particle size of 3.5 μm.

[0014] Preferably, the column temperature for the high-performance liquid chromatography detection is 30–60°C.

[0015] Preferably, the wavelength for detection by high performance liquid chromatography is 355 nm.

[0016] Preferably, the flow rate of the mobile phase for high performance liquid chromatography detection is 0.3 to 2 mL / min.

[0017] Preferably, the injection volume for high performance liquid chromatography detection is 10–50 μL.

[0018] Preferably, in the diluent, the volume ratio of isopropanol to other organic solvents is 95:5 to 60:40, and the volume ratio of water to organic phase is 10:90 to 40:60.

[0019] The diluent also includes an antioxidant, and the concentration of the antioxidant in the diluent is 1-15 g / L.

[0020] Preferably, the degreasing agent comprises at least one of transition metal sulfate, transition metal chloride, alkaline earth metal chloride, and alkaline earth metal sulfate;

[0021] The mass ratio of the retinoic acid preparation to the desiccant is 5–40:1.

[0022] Preferably, the step of mixing and extracting the retinoic acid preparation to be tested, the breaker and the diluent includes: shaking the retinoic acid preparation to be tested and the breaker until the suspension is dispersed, then adding a portion of the diluent for ultrasonic extraction, and adding the remaining diluent to make up the volume;

[0023] The concentration of retinoic acid in the sample solution to be tested is 50–150 μg / mL.

[0024] This invention first pre-treats the retinoic acid preparation to be tested: the preparation, a degelatinizing agent, and a diluent are mixed, and a solid-liquid separation operation is performed. The collected liquid component is the test sample solution. This pre-treatment method effectively removes matrix interference from the preparation. High-performance liquid chromatography (HPLC) is used to detect the test sample solution, enabling rapid qualitative and quantitative analysis of retinoic acid and seven related substances in the retinoic acid preparation. The peak shapes of retinoic acid and the seven related substances are good, the separation of each substance is high, the detection results are accurate, the detection limit is low, the quantitation limit is low, the detection sensitivity is high, and the stability is strong. The detection method provided by this invention overcomes the shortcomings of existing technologies in the detection of retinoic acid and its related substances in retinoic acid preparations, providing technical support for the testing and quality control of retinoic acid preparations. Attached Figure Description

[0025] Figure 1 This is the HPLC chromatogram of the system suitability solution in Example 1;

[0026] Figure 2 The above are HPLC chromatograms of the test solutions of retinoic acid-related substances in the diluent, blank preparation and multi-specification retinoic acid gel in Example 1.

[0027] Figure 3 This is the HPLC chromatogram of the limit-of-quantity solution in Example 2;

[0028] Figure 4 This is the HPLC chromatogram of the 100% accuracy solution in Example 2;

[0029] Figure 5 This is the HPLC chromatogram for the repeatability test in Example 2;

[0030] Figure 6 The HPLC chromatograms are for the stability study of the test solution in Example 2 (0h and 48h).

[0031] Figure 7 The HPLC chromatograms are of the system suitability solution and the commercially available semi-solid formulation (ointment / cream) test solution of retinoic acid related substances in Example 3.

[0032] Figure 8 Chromatograms of system-adaptive solutions for the determination of retinoic acid-related substances using different chromatographic columns in Comparative Example 2. Detailed Implementation

[0033] This invention provides a method for detecting retinoic acid and / or related substances in retinoic acid preparations, characterized by comprising the following steps:

[0034] The retinoic acid preparation to be tested, the gelling agent, and the diluent are mixed, and the solid and liquid components are separated, with the liquid component being the sample solution to be tested. The diluent includes water and an organic phase, the organic phase including isopropanol and other organic solvents, the other organic solvents including at least one of methyl tert-butyl ether, ethyl acetate, and tetrahydrofuran.

[0035] The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of retinoic acid and / or related substances;

[0036] The conditions for the high-performance liquid chromatography (HPLC) detection include: a Waters XSelect CSH Fluoro-Phenyl column and a Welch column. PAH column or Waters XSelect HSS PFP column; mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is 0.1–0.5 v / v% formic acid aqueous solution or 0.5–2 v / v% acetic acid aqueous solution, and mobile phase B is methanol, 1–10 v / v% methyl tert-butyl ether-methanol solution or 0.3–3 v / v% methyl tert-butyl ether-isopropanol solution; elution mode is isocratic elution or gradient elution; during isocratic elution, the volume ratio of mobile phase A to mobile phase B is 10:90–90. :10; The gradient elution program is as follows: 0–21 min, the volume fraction of mobile phase A is 80%; 21–30 min, the volume fraction of mobile phase A linearly decreases from 80% to 60%; 30–50 min, the volume fraction of mobile phase A linearly decreases from 60% to 30%; 50–55 min, the volume fraction of mobile phase A linearly increases from 30% to 80%; 55–70 min, the volume fraction of mobile phase A is 80%.

[0037] The related substances include one or more of isotretinoin, 9,13-cis-retinoic acid, 11,13-cis-retinoic acid, avitamin A, 9-cis-retinoic acid, all-trans-4-methoxyretinoic acid, and all-trans-5,6-epoxyretinoic acid.

[0038] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0039] This invention involves mixing the retinoic acid preparation to be tested, a degelatinizing agent, and a diluent, followed by solid-liquid separation, with the liquid component being the sample solution to be tested.

[0040] In this invention, the retinoic acid preparation to be tested is preferably a topical retinoic acid preparation, more preferably a retinoic acid semi-solid preparation, and may specifically be at least one of retinoic acid ointment and retinoic acid gel.

[0041] In this invention, the breaker preferably includes at least one selected from transition metal sulfates, transition metal chlorides, alkaline earth metal chlorides, and alkaline earth metal sulfates; more preferably, it is at least one selected from manganese sulfate, copper chloride, calcium chloride, barium chloride, and calcium sulfate, specifically at least one selected from barium chloride and calcium chloride. The breaker preferably includes at least one selected from anhydrous breaker and breaker hydrate. In this invention, the mass ratio of the retinoic acid preparation to the breaker is preferably 5–40:1, specifically 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, or 40:1.

[0042] In this invention, the diluent comprises water and an organic phase, wherein the organic phase comprises isopropanol and other organic solvents, and the other organic solvents include at least one selected from methyl tert-butyl ether, ethyl acetate, and tetrahydrofuran. In this invention, the volume ratio of isopropanol to other organic solvents in the diluent is preferably 95:5 to 60:40, specifically 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, or 60:40. In this invention, the organic phase may specifically be an isopropanol-methyl tert-butyl ether solution or an isopropanol-tetrahydrofuran solution, wherein the volume ratio of isopropanol to methyl tert-butyl ether in the isopropanol-methyl tert-butyl ether solution is preferably 80:20; and the volume ratio of isopropanol to tetrahydrofuran in the isopropanol-tetrahydrofuran solution is preferably 60:40. In this invention, the volume ratio of water to organic phase is preferably 10:90 to 40:60, and may specifically be 10:90, 15:85, 20:80, 25:75, 30:70, 35:65 or 40:60.

[0043] In this invention, the diluent preferably further includes an antioxidant, which preferably includes at least one of 2,6-di-tert-butyl-p-methylphenol, butylated hydroxyanisole, vitamin E, and preferably 2,6-di-tert-butyl-p-methylphenol; the concentration of the antioxidant in the diluent is preferably 1 to 15 g / L, specifically 1 g / L, 3 g / L, 5 g / L, 8 g / L, 10 g / L, 1 g / L, or 15 g / L.

[0044] In this invention, the preferred method of mixing and extracting the retinoic acid preparation to be tested, the breaker, and the diluent includes: shaking the retinoic acid preparation to be tested and the breaker until the suspension is dispersed, then adding a portion of the diluent for ultrasonic extraction, and finally adding the remaining diluent to make up the volume. In this invention, the volume of the diluent is preferably 40%–70% of the total volume of the diluent, specifically 40%, 45%, 50%, 55%, 60%, 65%, or 70%. In this invention, the ultrasonic extraction temperature is preferably room temperature (18–30°C); the ultrasonic extraction power is preferably 100–250W, specifically 100W, 120W, 150W, 180W, 200W, 220W, or 250W; and the ultrasonic extraction time is preferably 10–30 minutes, specifically 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

[0045] In this invention, the temperature for solid-liquid separation is preferably 2–8°C, specifically 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C. This invention does not impose any particular limitation on the solid-liquid separation; any solid-liquid separation method well-known to those skilled in the art can be used, such as filtration, vacuum filtration, or centrifugation, with centrifugation being more preferred. In this invention, the centrifugation speed is preferably 8000–15000 r / min, specifically 8000 r / min, 10000 r / min, 12000 r / min, or 15000 r / min; the centrifugation time is preferably 5–10 min, specifically 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0046] In this invention, the concentration of retinoic acid in the sample solution to be tested is preferably 50-150 μg / mL, specifically 50 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL or 150 μg / mL.

[0047] After obtaining the sample solution to be tested, the present invention performs high performance liquid chromatography on the sample solution to obtain the detection results of retinoic acid and / or related substances.

[0048] In this invention, the retinoic acid and related substances are shown in Table 1:

[0049] Table 1. Retinoic acid and related substances

[0050]

[0051] In this invention, the conditions for high-performance liquid chromatography (HPLC) detection include: a Waters XSelect CSHFluoro-Phenyl column and a Welch column. PAH column or Waters XSelect HSS PFP column; the column size is preferably 4.6 × 150 mm, and the packing particle size is preferably 3.5 μm; the column temperature is preferably 30–60℃, specifically 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, or 60℃; the mobile phase includes mobile phase A and mobile phase B; mobile phase A is a 0.1–0.5 v / v% formic acid aqueous solution or a 0.5–2 v / v% acetic acid aqueous solution, wherein the volume fraction of formic acid in the 0.1–0.5 v / v% formic acid aqueous solution can be specifically 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%; the volume fraction of acetic acid in the 0.5–2 v / v% acetic acid aqueous solution can be specifically... The mobile phase B is 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, or 2%; the mobile phase B is methanol, a 1-10 v / v% methyl tert-butyl ether-methanol solution, or a 0.3-3 v / v% methyl tert-butyl ether-isopropanol solution; the volume fraction of methyl tert-butyl ether in the 1-10 v / v% methyl tert-butyl ether-methanol solution can specifically be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%; the volume fraction of methyl tert-butyl ether in the 0.3-3 v / v% methyl tert-butyl ether-isopropanol solution can specifically be 0.3%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, or 1.8%. The elution rate is 2%, 2.2%, 2.5%, 2.8%, or 3%; the elution method is isocratic elution or gradient elution; during isocratic elution, the volume ratio of mobile phase A to mobile phase B is 10:90 to 90:10, specifically 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, or 90:10; the gradient elution program is as follows: 0–21 min, the volume fraction of mobile phase A is 80%; 21–30 min, the volume fraction of mobile phase A linearly decreases from 80% to 60%; 30–50 min, the volume fraction of mobile phase A decreases from 60% to... The volume fraction of mobile phase A linearly decreases to 30%; from 50 to 55 min, the volume fraction of mobile phase A linearly increases from 30% to 80%; from 55 to 70 min, the volume fraction of mobile phase A reaches 80%; the detection wavelength is preferably 355 nm; the mobile phase flow rate is preferably 0.3 to 2 mL / min, specifically 0.3 mL / min, 0.5 mL / min, 0.8 mL / min, 1 mL / min, 1.2 mL / min, 1.5 mL / min, 1.8 mL / min, or 2 mL / min; the injection volume is preferably 10 to 50 μL, specifically 10 μL, 20 μL, 30 μL, 40 μL, or 50 μL.

[0052] In this invention, the high-performance liquid chromatography detection preferably includes quantitative detection and / or qualitative detection.

[0053] In this invention, the qualitative detection step preferably includes: measuring the test sample solution and the mixed reference working solution under the conditions described above for ultra-high performance liquid chromatography detection, recording the chromatographic retention times of retinoic acid and / or related substances in the test sample solution and the mixed reference working solution, and determining that the corresponding compound is detected in the sample when a chromatographic peak (within ±2.5%) is detected in the test sample solution that is consistent with the retention time of retinoic acid and / or related substances in a mixed reference working solution is found.

[0054] In this invention, the preparation method of the mixed reference standard working solution preferably includes the following steps: accurately weighing reference standards RA, RA-A, RA-B, RA-C, RA-D, RA-F, and RA-G, dissolving them separately with diluent and diluting to a fixed volume to obtain stock solutions of each reference standard; accurately transferring each of the above-mentioned stock solutions of reference standards into the same volumetric flask, dissolving them with diluent and quantitatively diluting to obtain the mixed linear working solution. In this invention, the concentration of each reference standard in the stock solutions of reference standards is preferably 50–200 μg / mL, specifically 50 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 150 μg / mL, 180 μg / mL, or 200 μg / mL. In this invention, the concentration of a single reference standard in the mixed linear working solution is preferably 2.5 to 10 μg / mL, specifically 2.5 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL.

[0055] In this invention, the quantitative detection preferably includes a self-comparison method with a correction factor, and preferably includes the following steps: measuring the sample solution to be tested and the control solution according to the above-mentioned ultra-high performance liquid chromatography detection conditions, and recording the chromatogram; using the self-comparison method with a correction factor, calculating the content of retinoic acid and its related substances based on the peak area.

[0056] In this invention, the concentration of retinoic acid in the control solution is preferably 0.25–0.75 μg / mL, specifically 0.25 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, or 0.75 μg / mL. In this invention, the concentration of retinoic acid in the control solution is preferably 0.5% or 1% of the concentration of retinoic acid in the sample solution.

[0057] To further illustrate the present invention, the detection methods for retinoic acid and / or related substances in the retinoic acid preparations provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] In the following embodiments,

[0059] The main instrument consumables are as follows:

[0060] Liquid chromatography: The high-performance liquid chromatograph was a Thermo Fisher Vanquish Core high-performance liquid chromatograph, and the detector was a UV detector; the chromatography workstation used Chromeleon 7.2.10 software;

[0061] Electronic balances: BCA224I-1OCN, Quintix65-1CN, Sartorius;

[0062] Comfort I ultrapure water system, Sartorius;

[0063] 5425R Low-Temperature High-Speed ​​Centrifuge, Eppendorf;

[0064] M5800H-C Ultrasonic Cleaner, Branson.

[0065] Information on retinoic acid impurity reference standards is shown in Table 2.

[0066] Table 2 Information on Retinoic Acid Impurity Reference Standards

[0067] name factory Retinoic acid (RA) reference standard China Food and Drug Inspection Institute Retinoic acid impurity A (RA-A) China Food and Drug Inspection Institute Retinoic acid impurity B (RA-B) QCC Retinoic acid impurity C (RA-C) QCC Retinoic acid impurity D (RA-D) QCC Retinoic acid impurity F (RA-F) QCC Retinoic acid impurity G (RA-G) QCC

[0068] Example 1

[0069] Detection of retinoic acid and related substances in multi-specification retinoic acid gels

[0070] 1.1 Experimental Procedure:

[0071] 1.1.1 Preparation of related formulations

[0072] Table 3. Retinoic acid gel and blank gel formulations (mass fraction)

[0073]

[0074] Preparation of homemade retinoic acid gel (formula shown in Table 3): Weigh 60 wt% of the prescribed amount of water, add sodium methylparaben and sodium propylparaben, and stir to dissolve. Add carbomer and stir at high speed (200 rpm) for 5 min to disperse it evenly; then stir at low speed (50 rpm) for 50 min to hydrate the carbomer and obtain the aqueous phase; add retinoic acid, butylated hydroxytoluene, and lauryl cyanide. The ketone and propylene glycol were stirred until a homogeneous oil phase was formed. The aqueous phase was added to the oil phase, and the mixture was homogenized for 3 minutes at 5000 rpm using a high-shear homogenizer. NaOH was added to adjust the pH to 5.3 ± 0.2, and the remaining water was added. The mixture was then mechanically stirred at low speed for 30 minutes to obtain the self-made retinoic acid gel. The gel was then filled into aluminum-plastic composite tubes.

[0075] Preparation of blank gel (formulation shown in Table 3): The only difference from the preparation steps of homemade retinoic acid gel is that retinoic acid is not added.

[0076] 1.1.2 Solution Preparation

[0077] (1) Preparation of diluent: 0.2 g of di-tert-butyl-p-methylphenol, 80 mL of isopropanol and 20 mL of methyl tert-butyl ether were sonicated until completely dissolved and shaken to obtain solvent A; water was added and mixed evenly to obtain diluent. The volume ratio of water to solvent A was 20:80.

[0078] (2) Preparation of system adaptability solution: Dissolve and quantitatively dilute the retinoic acid, RA-A, RA-B, RA-C, RA-D, RA-F and RA-G impurity reference standards with diluent to obtain a system adaptability solution with a retinoic acid concentration of 100 μg / mL and the concentration of each of the other impurity reference standards of 0.5 μg / mL.

[0079] (3) Preparation of the test solution: Accurately weigh the self-made retinoic acid gel (retinoic acid specifications 0.01%, 0.025%, and 0.05%), equivalent to 1 mg of retinoic acid, and place it in a 20 mL volumetric flask. Add 0.3 g of barium chloride, shake until the suspension is dispersed, add diluent to 40–70% of the volumetric flask volume, sonicate for 20 min, and finally dilute to the mark with diluent to obtain a sample solution containing 50 μg / mL of retinoic acid. Centrifuge at 4℃ and 12000 r / min for 5 min, and take the supernatant as the test solution. Prepare 6 parallel aliquots of the test solution.

[0080] (4) Preparation of control solution: Accurately measure 0.5 mL of the test solution, place it in a 100 mL volumetric flask, add diluent to make up to the mark, and shake well.

[0081] (5) Preparation of retinoic acid reference solution: Dissolve retinoic acid reference standard in diluent and quantify to obtain 50 μg / mL retinoic acid reference solution, which is used to evaluate the extraction recovery rate of the main component by the extraction method.

[0082] (6) Preparation of single-component positioning solutions: Accurately weigh RA-A, RA-B, RA-C, RA-D, RA-F, RA-G and RA reference standards, dissolve them in methanol and dilute them quantitatively to obtain stock solutions of each impurity reference standard with a concentration of 0.1 mg / mL; accurately measure 1 mL of the above impurity reference standard stock solution into different 10 mL volumetric flasks, add diluent to the mark, shake well, and use as single-component positioning solutions.

[0083] (7) Preparation of blank preparation sample solution: Accurately weigh 10g of blank gel (the sample weight is equivalent to the 0.01% specification), place it in a 20mL volumetric flask, add 0.3g of barium chloride, shake until the suspension is dispersed, add diluent to 40-70% of the volumetric flask volume, sonicate for 20min, and finally dilute to the mark with diluent to obtain a sample solution containing 50μg / mL of retinoic acid. Centrifuge at 4℃ and 12000r / min for 5min, and take the supernatant blank preparation sample solution.

[0084] 1.1.3 HPLC Chromatographic Conditions

[0085] The chromatographic column was a Waters XSelect HSS PFP, with dimensions of 4.6 × 150 mm and a packing particle size of 3.5 μm; mobile phase A was 0.1 v / v% formic acid aqueous solution; mobile phase B was methanol; the elution method was isocratic elution, with a mobile phase:mobile phase B volume ratio of 20:80; the detection wavelength was 355 nm; the mobile phase flow rate was 1.0 mL / min; the column temperature was 35 °C; and the injection volume was 35 μL.

[0086] 1.1.4 Determination Method

[0087] (1) Perform HPLC analysis on the system adaptability solution, test solution, control solution and retinoic acid reference solution respectively, and record the chromatograms.

[0088] (2) The extraction and recovery rate of retinoic acid in compound multi-specification (0.01-0.05%) retinoic acid gel was calculated by peak area using the external standard method.

[0089] (3) The known impurity content of each specification of retinoic acid gel was calculated by peak area using the self-comparison method with correction factor.

[0090] 1.2 Results and Conclusions

[0091] (1) The recovery rate test results are shown in Table 4. As can be seen from Table 4, the sample pretreatment method (mixed extraction of retinoic acid preparation, gel breaking agent and diluent) involved in this invention can effectively extract retinoic acid components from retinoic acid gels of various specifications, with an overall recovery rate of 97.07-106.57% and RSD≤0.83%.

[0092] Table 4. Recovery results of retinoic acid extraction from different specifications of retinoic acid gel (peak area unit: mAU×min; recovery rate unit: %)

[0093]

[0094] Note: "NA" in each table indicates that it is not applicable.

[0095] (2) System suitability solution chromatograms are shown in [reference needed]. Figure 1 The resolution of the chromatographic peaks of retinoic acid and various impurities is shown in Table 5. The results indicate that baseline separation can be achieved for each substance under these chromatographic conditions.

[0096] Table 5. Resolution of chromatographic peaks of retinoic acid and various impurities

[0097]

[0098] Resolution (after) refers to the resolution between the analyte and the adjacent chromatographic peaks after the chromatographic peak.

[0099] (3) The known impurity content of each substance was calculated based on peak area using a self-comparison method with correction factors. Chromatograms of each specification of retinoic acid gel are shown below. Figure 2 As shown in Table 6, the test results for related substances are as follows. The results indicate that the detection method provided by this invention, when used for the detection of retinoic acid gel formulations, has no unidentified interfering peaks and is suitable for impurity control in retinoic acid gel formulations.

[0100] Table 6. Detection results of related substances in various specifications of retinoic acid gel.

[0101]

[0102]

[0103] Note: "3 months" refers to the test results after 3 months of stability testing under conditions of 30±2℃ and RH of 65±5%.

[0104] Example 2

[0105] Methodological validation results of retinoic acid related substances

[0106] Experimental objective: To validate the preferred analytical method and confirm its performance.

[0107] Diluent: The diluent prepared in Example 1.

[0108] HPLC chromatographic conditions: Same as in Example 1.

[0109] 2.1 Specificity

[0110] Monitor the interference of blank solvents and gel excipients on the components to be tested.

[0111] Blank solvent: diluent.

[0112] Blank excipient test solution: Similar to the retinoic acid gel in Example 1, there are multiple specifications. In this experiment, the amount of blank excipient added is equivalent to the sample amount of 0.01% self-made retinoic acid gel (smallest specification). Weigh the blank excipient accurately and place it in a 20mL volumetric flask. Add 0.3g of calcium chloride, shake until the suspension is dispersed, then add diluent to 40-70% of the volumetric flask volume. Extract by sonication for 20min, and dilute to the mark with diluent. Centrifuge at 4℃ and 12000r / min for 5min, and take the supernatant as the blank excipient test solution.

[0113] The blank solvent and blank excipient test solutions were analyzed by HPLC, and the chromatograms were recorded. The results showed that the detection method of the present invention has excellent specificity, the blank solvent and excipients do not interfere with the determination of retinoic acid and its related substances, and the resolution between impurities and the main component is greater than 1.5.

[0114] 2.2 Sensitivity Test

[0115] Limit of Quantitation (LOQ) Solutions: Dissolve and dilute the reference standards for each impurity (retinoic acid, RA-A, RA-B, RA-C, RA-D, RA-F, and RA-G) separately with diluent to prepare stock solutions with a concentration of 100 μg / mL. Dilute each stock solution with diluent to concentrations of 15 ng / mL, 15 ng / mL, 25 ng / mL, 20 ng / mL, 20 ng / mL, 10 ng / mL, and 4 ng / mL for retinoic acid, RA-A, RA-B, RA-C, RA-D, RA-F, and RA-G, respectively, to obtain the LQ solutions. Prepare six parallel aliquots.

[0116] Each limit of quantitation was determined by HPLC, and the limit of quantitation was determined by the amount injected into the chromatograph at a signal-to-noise ratio (S / N) of 10:1. Results are shown below. Figure 3 See Table 7.

[0117] When the concentration of the retinoic acid test solution is 50 μg / mL, the quantitation limits of each component are 4.529–25.14 ng / mL, which is equivalent to 0.0091–0.050% of the test solution concentration. The S / N ratios are all greater than 10, indicating that the detection method provided by this invention has good sensitivity for determining retinoic acid and related substances.

[0118] Table 7 Results of Limit of Quantitation Tests (Peak area unit: mAU×min)

[0119]

[0120] Limit of Detection (LOD) Solutions: Take appropriate amounts of each LOD solution and serially dilute with diluent to concentrations of retinoic acid, RA-A, RA-B, RA-C, RA-D, RA-F, and RA-G of 5 ng / mL, 5 ng / mL, 8 ng / mL, 7 ng / mL, 7 ng / mL, 3 ng / mL, and 1 ng / mL, respectively, to obtain the LOD solutions for each component. Perform HPLC analysis on each LOD solution. The LOD was determined using the injection volume at a signal-to-noise ratio (S / N) of 3:1. The results are shown in Table 8.

[0121] The results showed that when the concentration of retinoic acid test sample was 50 μg / mL, the detection limits of each substance were 1.510–8.380 ng / mL, which is equivalent to 0.0030–0.017% of the test sample concentration. The S / N ratios were all greater than 3, indicating good sensitivity.

[0122] Table 8. Detection Limit Test Results

[0123] name Concentration (ng / mL) Equivalent to the percentage of the test sample concentration (%) S / N RA-G 1.510 0.0030 9.5 RA-F 3.673 0.0073 9.4 RA-C 6.900 0.014 10.6 RA-A 5.297 0.011 10.6 RA-B 8.380 0.017 9.5 RA-D 7.520 0.015 8.8 RA 5.329 0.011 6.1

[0124] 2.3 Accuracy Test

[0125] The determination of impurities was carried out by adding known amounts of the blank excipients in the formulation. The impurities included RA-A, RA-B, RA-C, RA-D, RA-F, and RA-G. The accuracy of the test for unknown impurities was evaluated by adding retinoic acid. This product is a multi-specification retinoic acid gel. In this experiment, the amount of blank excipient added was set to be equivalent to the minimum specification (0.01%) sample amount in Example 1.

[0126] Preparation of accuracy mixed working solutions: Weigh RA and each impurity reference standard, add diluent to dissolve and quantitatively dilute to prepare accuracy mixed working solutions with RA-A concentration of 25 μg / mL, RA-B, RA-C, RA-D, RA-F, RA-G and RA concentration of 5 μg / mL.

[0127] Preparation of background solution: Weigh the blank preparation (sample amount equivalent to the smallest specification of retinoic acid), accurately place it in a 20mL volumetric flask, add 0.3g of calcium chloride, shake until the suspension is dispersed, add diluent to 40-70% of the volumetric flask volume, sonicate for 20min, add diluent to make up to the mark, centrifuge at 4℃ and 12000r / min for 5min, and take the supernatant as the background solution.

[0128] Preparation of the 50% accuracy solution: Using the method described in the "Preparation of Background Solution" section, prepare a background blank excipient suspension until dispersed. Accurately measure 1 mL of the accuracy working solution and place it in an identical volumetric flask. Add an appropriate amount of diluent to 40–70% of the volumetric flask volume. Sonicate for 20 min, add diluent to the mark, and centrifuge at 4℃ and 12000 r / min for 5 min. Take the supernatant as the 50% accuracy solution. Prepare three parallel aliquots.

[0129] Preparation of 100% accuracy solution: Using the method described in "Preparation of Background Solution", prepare a background blank excipient suspension until dispersed. Accurately measure 2 mL of the accuracy mixed working solution and place it in an identical volumetric flask. Add an appropriate amount of diluent, sonicate for 20 min, and dilute to the mark with diluent. Centrifuge at 4℃ and 12000 r / min for 5 min. Take the supernatant as the 100% accuracy solution. Prepare three parallel aliquots.

[0130] Preparation of 150% accuracy solution: Using the method described in "Preparation of Background Solution", prepare a background blank excipient suspension until dispersed. Accurately measure 3 mL of the accuracy mixed working solution and place it in an identical volumetric flask. Add an appropriate amount of diluent, sonicate for 20 min, and dilute to the mark with diluent. Centrifuge at 4℃ and 12000 r / min for 5 min. Take the supernatant as the 150% accuracy solution. Prepare three parallel aliquots.

[0131] Each accuracy solution was analyzed by HPLC. In this example, the recovery rate % = (measured amount - background amount) / added amount × 100%. Results are shown below. Figure 4 (100% accuracy solution) and Table 9. The results show that, using the detection method provided by this invention to test retinoic acid gel and its related substances, the average accuracy of each component is in the range of 92.92% to 99.65%, and the RSD is no greater than 3.42%, indicating that the detection method provided by this invention has good accuracy.

[0132] Table 9 Accuracy Test Results

[0133]

[0134] 2.4 Linear Experiment

[0135] Preparation of stock solutions for each reference standard: Accurately weigh RA, impurity RA-A, RA-B, RA-C, RA-D, RA-F, and RA-G reference standards, dissolve and dilute with diluent to obtain stock solutions of each reference standard with a concentration of 100 μg / mL.

[0136] Mixed working solutions of reference standards: Accurately transfer each of the above-mentioned stock solutions of reference standards into the same volumetric flask, dissolve and quantitatively dilute with diluent to prepare a mixed linear working solution with a concentration of 5 μg / mL for each reference standard. Accurately measure each mixed linear working solution and dilute with diluent to prepare a series of mixed linear solutions of reference standards at different concentrations. The concentrations of each reference standard (RA-A, RA-B, RA-C, RA-D, RA-F, RA-G, RA) are 0.025 μg / mL, 0.05 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1.25 μg / mL, 2.5 μg / mL, and 5 μg / mL, respectively.

[0137] The mixed reference standard working solution was analyzed by HPLC. For each component within the detection concentration range, a linear regression was performed with concentration C as the x-axis (X-value) and peak area as the y-axis, with a weight of 1 / C. 2 Correlation coefficient r 2 The response factor (RSD) should be greater than 0.99, and the response factor RSD should not exceed 10%. The results are shown in Table 10. The results indicate that the detection method provided by this invention has a wide linear range and good linearity.

[0138] Table 10 Summary of Linearity Observation Results

[0139] name Linear range (μg / mL) Linear equations <![CDATA[Coefficient of correlation r 2 > Response factor RSD (%) RA-G 0.0328～6.568 Y = 3.791X + 0.0071 0.9931 2.05 RA-F 0.0366～7.321 Y = 4.473X + 0.0780 0.9997 4.66 RA-C 0.0310～6.205 Y = 1.793X - 0.0002 0.9996 2.55 RA-A 0.0255～5.100 Y = 3.815X - 0.0048 0.9998 1.99 RA-B 0.0280～5.594 Y = 3.829X - 0.0043 0.9994 2.45 RA-D 0.0372～7.438 Y = 3.841X - 0.0051 0.9998 2.26 RA 0.0253～5.067 Y = 5.252X - 0.0019 0.9994 1.69

[0140] 2.5 Precision Experiment

[0141] 2.5.1 Repeatability

[0142] The self-made retinoic acid gel (retinoic acid specifications of 0.01% and 0.05%) from Example 1 was selected, and test solutions and control solutions were prepared in parallel, six times each, according to section 1.1.1 of Example 1. Each solution was analyzed by HPLC, and the results are as follows: Figure 5 As shown.

[0143] In the low-specification (0.01%) test solution, unknown impurity 1 (UNK 1) was detected, with an average detection amount of 0.065% and an RSD of 7.75% in 6 samples; isomer impurity RA-A was detected, with an average detection amount of 0.169% and an RSD of 5.94%; the average total impurity (excluding RA-A) was 0.122% and the RSD was 5.22%.

[0144] In the high-specification (0.05%) test solution, unknown impurity 1 (UNK 1) was detected, with an average detection amount of 0.079% and an RSD of 5.65% in 6 samples; isomer impurity RA-A was detected, with an average detection amount of 0.117% and an RSD of 3.61%; isomer impurity RA-G was detected, with an average detection amount of 0.070% and an RSD of 0.93%; the average total impurity (excluding RA-A) was 0.196% and the RSD was 2.67%.

[0145] 2.5.2 Intermediate Precision

[0146] In the same laboratory, at different times, different analysts used different high-performance liquid chromatographs (Shimadzu LC-20A and Thermo Fisher Vanquish Core) to collect the same concentration of test sample within the specified range. Six determination results were compared and analyzed with the repeatability test results. The results are as follows:

[0147] In the low-specification (0.01%) test solution, unknown impurity 1 (UNK 1) was detected, with an average detection amount of 0.062% and an RSD of 7.50% for 12 samples; isomer impurity RA-A was detected, with an average detection amount of 0.193% and an RSD of 8.94%; the average total impurity (excluding RA-A) was 0.110% and the RSD was 2.44%.

[0148] In the high-specification (0.05%) test solution, unknown impurity 1 (UNK 1) was detected, with an average detection amount of 0.076% and an RSD of 5.67% for 12 samples; isomer impurity RA-A was detected, with an average detection amount of 0.132% and an RSD of 8.61%; isomer impurity RA-G was detected, with an average detection amount of 0.067% and an RSD of 5.19%; the average total impurity amount (excluding RA-A) was 0.191% and the RSD was 3.74%.

[0149] The detection method provided by this invention can be used to test retinoic acid and related substances in various specifications of gels. It has good detection ability for potential isomer impurities and degradation impurities, and the method has good repeatability and precision.

[0150] 2.6 Solution stability experiment

[0151] Referring to section 2.5 Precision Test, test solutions and control solutions of self-made retinoic acid gel of different specifications were prepared and incubated at 10℃ for 0h, 5h, 10h, 15h, 20h, 27h, 36h, and 48h, respectively, before HPLC detection. The results are shown in [Figure 1]. Figure 6 ,Depend on Figure 6 It can be seen that:

[0152] (1) When the low-specification (0.01%) test solution was placed at 10℃ for 48h, an unknown impurity 1 was detected, with an average detection amount of 0.070%. The maximum difference between the samples measured at 8 time points was 0.016%, and the RSD was 7.74%. The isomer impurity RA-A was detected, with an average detection amount of 0.167%. The maximum difference between the measurements was 0.003%, and the RSD was 0.79%. The average amount of total impurities detected (excluding RA-A) was 0.118%. The maximum difference between the measurements was 0.024%, and the RSD was 5.91%.

[0153] (2) When the high-specification (0.05%) test solution was placed at 10℃ for 48h, the following were observed: Unknown impurity 1 was detected with an average detection amount of 0.088%, the maximum difference between the samples at 8 time points was 0.017%, and the RSD was 5.67%; isomer impurity RA-A was detected with an average detection amount of 0.122%, the maximum difference between the samples was 0.009%, and the RSD was 2.16%; isomer impurity RA-G was detected with an average detection amount of 0.074%, the maximum difference between the samples was 0.004%, and the RSD was 1.82%; the average amount of total impurities detected (excluding RA-A) was 0.209%, the maximum difference between the samples was 0.012%, and the RSD was 2.16%.

[0154] (3) The control solution was placed at 10℃ for 48h, and the RSD of the peak area of ​​the main peak was ≤1.71% for the peak area of ​​the 0h peak.

[0155] The results showed that both the test solution and the reference solution had good stability after being placed at 10℃ for 48 hours.

[0156] Example 3

[0157] Testing for retinoic acid-related substances in commercially available semi-solid preparations (ointments / creams)

[0158] Objective: To demonstrate the feasibility of the developed analytical method (gradient elution). Under the described chromatographic conditions, the various impurities were well separated.

[0159] 3.1 Experimental Procedure:

[0160] 3.1.1 Solution Preparation

[0161] (1) Preparation of diluent

[0162] Dissolve 0.5 g of di-tert-butyl-p-methylphenol, 60 mL of isopropanol, and 40 mL of tetrahydrofuran by sonication, and shake well to obtain diluent A. Mix water and diluent A in a volume ratio of 35:65 to obtain diluent B.

[0163] (2) Preparation of system adaptability solution

[0164] Take the reference standards for each impurity, namely retinoic acid, RA-A, RA-B, RA-C, RA-D, RA-F and RA-G, dissolve them in diluent B and dilute quantitatively to prepare a system adaptability solution containing 100 μg / mL retinoic acid and 0.5 μg / mL of other reference standards.

[0165] (3) Preparation of test solution

[0166] The information on the commercially available retinoic acid semi-solid dosage form tested is as follows:

[0167] Commercially available cream A: Baoyining, 0.05% specification, Guangdong Xiangshantang Pharmaceutical Co., Ltd.

[0168] Commercially available cream B: Youweijia, 0.025%, Shandong Liangfu Pharmaceutical Co., Ltd.

[0169] Commercially available cream C: Retin-A, 0.05%, VALEANT BERMUDA.

[0170] Weigh out 1 mg of commercially available retinoic acid semi-solid preparation (retinoic acid specification 0.025-0.05%), accurately place it in a 20 mL volumetric flask, add 0.3 g of calcium chloride, shake until the suspension is dispersed, add 15 mL of diluent A, sonicate for 20 min, and finally dilute to the mark with diluent A to prepare a sample solution containing 50 μg / mL retinoic acid. Centrifuge at 4℃ and 12000 r / min for 5 min, and take the supernatant as the test solution.

[0171] (4) Preparation of control solution

[0172] Accurately measure 0.5 mL of the test solution and place it in a 100 mL volumetric flask. Add diluent B to the mark and shake well.

[0173] (5) Preparation of reference solution

[0174] The retinoic acid reference standard was dissolved and quantified with diluent B to obtain a 50 μg / mL retinoic acid reference standard solution, which was used to evaluate the extraction recovery rate of the main component by the extraction method.

[0175] (6) Preparation of positioning solution

[0176] Take appropriate amounts of RA-A, RA-B, RA-C, RA-D, RA-F, RA-G and RA reference standards, accurately weigh them, dissolve them in methanol and quantitatively dilute them to prepare solutions containing approximately 0.1 mg per ml, as stock solutions of each impurity reference standard; accurately measure 1 ml of the above solutions into different 10 ml volumetric flasks, add diluent B to the mark, shake well, and use as single-component positioning solutions.

[0177] 3.1.2 HPLC chromatographic conditions:

[0178] The chromatographic column was Welch. PAH, with specifications of 4.6×150mm and filler particle size of 3μm;

[0179] Mobile phase A was a 2.0 v / v % aqueous acetic acid solution; mobile phase B was methanol; the detection wavelength was 355 nm.

[0180] The mobile phase flow rate was 0.8 mL / min; the column temperature was 30 °C; the injection volume was 35 μL; the elution method was gradient elution, and the gradient elution program is shown in Table 11.

[0181] Table 11 Elution Procedure

[0182] Time / min Volume fraction of mobile phase A / % Mobile phase B volume fraction / % 0 80 20 21 80 20 30 60 40 50 30 70 55 80 20 70 80 20

[0183] 3.1.3 Determination Method

[0184] (1) Perform HPLC analysis on the system adaptability solution, test solution and control solution respectively, and record the chromatograms.

[0185] (2) The extraction recovery rate of retinoic acid in commercially available semi-solid preparations (ointments / creams) of various specifications (0.01-0.05%) was calculated by peak area using the external standard method.

[0186] (3) The known impurity content of each commercially available preparation was calculated by peak area using the self-comparison method with correction factors.

[0187] 3.2 Results and Conclusions

[0188] (1) The test results are shown in Table 12 and Figure 7 The results showed that the sample pretreatment method of the present invention can effectively extract the retinoic acid component from commercially available retinoic acid semi-solid preparations, with an overall recovery rate of 97.68-102.97% and RSD≤1.52%.

[0189] Table 12. Recovery results of retinoic acid extraction from commercially available retinoic acid semi-solid preparations (peak area unit: mAU×min; recovery rate unit: %)

[0190]

[0191]

[0192] (2) System suitability: The resolution of retinoic acid and various impurity peaks in the solution chromatograms is shown in Table 13. The results indicate that baseline separation can be achieved for each substance under these chromatographic conditions.

[0193] Table 13 Resolution of retinoic acid and various impurity chromatographic peaks

[0194]

[0195] (3) The known impurity content of each commercially available formulation was calculated by peak area using a self-comparison method with correction factors. Chromatograms of retinoic acid gels at various specifications are shown below. Figure 2 As shown in Table 14, the impurity results indicate that the detection method provided by this invention is suitable for the detection of impurities in commercially available retinoic acid creams.

[0196] Table 14. Impurity results for various commercially available formulations.

[0197] name Commercially available cream A Commercially available cream B Commercially available cream C RA-A 0.350 0.587 0.465 RA-B ND ND ND RA-C ND ND ND RA-D 0.089 0.124 0.104 RA-F ND ND ND RA-G 0.120 0.088 ND Other single-mixed 0.090 0.056 0.077 Total impurities except RA-A 0.345 0.268 0.251

[0198] Note: "ND" indicates not detected.

[0199] Comparative Example 1

[0200] Determination of retinoic acid-related substances using methods listed in the European Pharmacopoeia

[0201] Experimental objective: To evaluate the impurity separation and detection capabilities of existing conventional analytical methods, and to illustrate the advantages of the method of the present invention in comparison.

[0202] 4.1 Experimental Procedure:

[0203] 4.1.1 Solution Preparation

[0204] (1) Solvent: Isopropanol and methanol are mixed at a volume ratio of 1:1.

[0205] (2) Preparation of impurity localization solution

[0206] Stock solutions for each impurity: Accurately weigh each reference standard RA-A, RA-B, RA-C, RA-D, RA-F, and RA-G, dissolve and dilute them in methanol to prepare solutions with a concentration of 100 μg / mL for each reference standard, which are used as stock solutions for impurities RA-A, RA-B, RA-C, RA-D, RA-F, and RA-G.

[0207] Isomer impurity A, B, and D positioning solutions: Accurately transfer 1 mL of the stock solution of impurity reference standards RA-A, RA-B, and RA-D into different 10 mL volumetric flasks, dilute to the mark with solvent, and shake well.

[0208] (3) Preparation of system adaptability solution

[0209] Measure 2.5 mL of RA-A reference stock solution and 1 mL each of RA-B, RA-C, RA-D, RA-F, and RA-G reference stock solutions, place them in a 100 mL volumetric flask, add 10 mg of retinoic acid reference standard, dissolve and dilute to the mark with solvent, and shake well.

[0210] 4.1.2 HPLC Chromatographic Conditions (European Pharmacopoeia Method): The chromatographic column was a YMC Hydrosphere C18, with dimensions of 4.6 mm × 150 mm and a packing particle size of 3 μm; the mobile phase was a water:methanol:glacial acetic acid mixture with a volume ratio of 225:770:5, with isocratic elution; the detection wavelength was 355 nm; the mobile phase flow rate was 1.0 mL / min; the column temperature was 40 °C; and the injection volume was 10 μL.

[0211] 4.1.3 Determination Method

[0212] The impurity localization solution and the system suitability solution were analyzed by HPLC, and the chromatograms were recorded.

[0213] 4.2 Results and Conclusions

[0214] The test results are shown in Table 15. The results indicate that under the relevant substance chromatographic conditions, impurity B overlaps with impurity A, and the resolution between impurity D and the main component retinoic acid is less than 1.5, failing to achieve baseline separation. This indicates that the method is not suitable for the detection of impurities B and D.

[0215] Table 15 shows the localization results of each component in the determination method of retinoic acid-related substances included in EP.

[0216]

[0217]

[0218] Comparative Example 2

[0219] Column screening

[0220] Experimental objective: To conduct tests using other commonly used C18 chromatographic columns and compare their performance to demonstrate the appropriateness of the selected column.

[0221] 5.1 Test Procedure

[0222] 5.1.1 Solution Preparation

[0223] (1) Diluent: Same as in Example 1.

[0224] (2) Preparation of system adaptability solution: Same as in Example 1.

[0225] 5.1.2 HPLC Chromatographic Conditions

[0226] Column 1: Agilent ZORBAX Extend C18, 4.6×250mm, with a packing particle size of 5μm;

[0227] Column 2: Waters Xbridge BEH C18, 4.6×150mm, with a packing particle size of 3.5μm;

[0228] Column 3: ACE Generix 3C18, 4.6×150mm, with a particle size of 3.5μm;

[0229] The remaining chromatographic conditions were the same as in Example 1.

[0230] 5.1.3 Determination Method

[0231] Precisely measured solutions suitable for the system were analyzed by HPLC.

[0232] 5.2 Results and Conclusions

[0233] The test results are as follows Figure 8 As shown in Table 16, the results indicate that when using chromatographic columns 1, 2, and 3, and eluting with the optimized mobile phase conditions described in Example 1, the isomer impurities RA-B and RA-A were difficult to separate, failing to achieve baseline separation. Specifically, when using columns 1 and 3, RA-B and RA-A almost overlapped into a single peak; when using column 2, the resolution between RA-B and RA-A was only 1.22, failing to meet baseline separation (resolution ≥ 1.5). When using column 2 and the optimized mobile phase conditions described in Example 1, the resolution between the isomer impurities RA-C and RA-A was only 0.99, also failing to achieve baseline separation. These test results suggest that the three different specifications of C18 columns used in this experiment all have certain separation defects when used for the analysis of retinoic acid-related substances, failing to meet the requirement of complete separation of isomer impurities.

[0234] Table 16 Separation results of each component during column optimization.

[0235]

[0236]

[0237] Note: " / " indicates that the current chromatographic peak is not separated from the subsequent chromatographic peaks and no relevant data can be obtained; "NA" indicates that it is not applicable.

[0238] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting retinoic acid and / or related substances in a retinoic acid preparation, characterized in that, Includes the following steps: The retinoic acid preparation to be tested, the gelling agent, and the diluent are mixed and extracted, followed by solid-liquid separation. The liquid component is the sample solution to be tested. The diluent includes water and an organic phase. The organic phase includes isopropanol and other organic solvents. The other organic solvents include at least one of methyl tert-butyl ether, ethyl acetate, and tetrahydrofuran. The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of retinoic acid and / or related substances; The conditions for the high-performance liquid chromatography (HPLC) detection include: a Waters XSelect CSH Fluoro-Phenyl column and a Welch column. PAH column or Waters XSelect HSS PFP column; mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is 0.1–0.5 v / v% formic acid aqueous solution or 0.5–2 v / v% acetic acid aqueous solution, and mobile phase B is methanol, 1–10 v / v% methyl tert-butyl ether-methanol solution or 0.3–3 v / v% methyl tert-butyl ether-isopropanol solution; elution mode is isocratic elution or gradient elution; during isocratic elution, the volume ratio of mobile phase A to mobile phase B is 10:90–90. :10; The gradient elution program is as follows: 0–21 min, the volume fraction of mobile phase A is 80%; 21–30 min, the volume fraction of mobile phase A linearly decreases from 80% to 60%; 30–50 min, the volume fraction of mobile phase A linearly decreases from 60% to 30%; 50–55 min, the volume fraction of mobile phase A linearly increases from 30% to 80%; 55–70 min, the volume fraction of mobile phase A is 80%. The related substances include one or more of isotretinoin, 9,13-cis-retinoic acid, 11,13-cis-retinoic acid, avitamin A, 9-cis-retinoic acid, all-trans-4-methoxyretinoic acid, and all-trans-5,6-epoxyretinoic acid.

2. The detection method according to claim 1, characterized in that, During the isocratic elution process, the volume ratio of mobile phase A to mobile phase B is 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, or 90:

10.

3. The detection method according to claim 1, characterized in that, The chromatographic column has a size of 4.6×150mm and a packing particle size of 3.5μm.

4. The detection method according to claim 1 or 3, characterized in that, The column temperature for the high-performance liquid chromatography detection is 30–60 °C.

5. The detection method according to claim 1, characterized in that, The wavelength for detection by high-performance liquid chromatography is 355 nm.

6. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase for the high-performance liquid chromatography detection is 0.3–2 mL / min.

7. The detection method according to claim 1, characterized in that, The injection volume for high performance liquid chromatography detection is 10–50 μL.

8. The detection method according to claim 1, characterized in that, In the diluent, the volume ratio of isopropanol to other organic solvents is 95:5 to 60:40, and the volume ratio of water to organic phase is 10:90 to 40:

60. The diluent also includes an antioxidant, and the concentration of the antioxidant in the diluent is 1-15 g / L.

9. The detection method according to claim 1, characterized in that, The de-gelling agent includes at least one of transition metal sulfates, transition metal chlorides, alkaline earth metal chlorides, and alkaline earth metal sulfates. The mass ratio of the retinoic acid preparation to the desiccant is 5–40:

1.

10. The detection method according to claim 1, 8, or 9, characterized in that, The step of mixing and extracting the retinoic acid preparation to be tested, the breaker and the diluent includes: shaking the retinoic acid preparation to be tested and the breaker until the suspension is dispersed, then adding a portion of the diluent and ultrasonically extracting, and then adding the remaining diluent to make up the volume; The concentration of retinoic acid in the sample solution to be tested is 50–150 μg / mL.

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