Method for detecting cyclosporine and related substances of oil-in-water emulsion of cyclosporine and application of cyclosporine and related substances

Abstract

The invention provides a cyclosporine bulk drug and a detection method of related substances of oil-in-water emulsion thereof, the method adopts an octadecyl silane bonded silica gel chromatographic column (the front of the column is connected with a stainless steel tube), a mobile phase A is a mixed solution of acetonitrile, a water phase, tert-butyl methyl ether and phosphoric acid in a ratio of 450-480: 490-540: 25-45: 1, and the water phase contains 0.1-0.4% of an ion pair reagent; a mobile phase B is a mixed solution of acetonitrile and 0.2% phosphoric acid water in a ratio of 70: 30-80: 20, and the cyclosporine and five impurities (impurity U, impurity H, impurity dihydroA, impurity D and impurity G) thereof can be distinguished and detected at the same time through specific gradient elution. The method has the advantages of strong specificity, high sensitivity and good repeatability, and can effectively control the content of related substances in the cyclosporine oil-in-water emulsion. The method can be used for detecting the cyclosporine ophthalmic preparation containing the teloxacin and the medium chain triglyceride.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting related substances in cyclosporine raw material and its oil-in-water emulsion, and its application. Background Technology

[0002] Cyclosporin, also known as cyclosporin A or cyclosporine A, with CAS number 59865-13-3 (i.e., Cyclosporin A or Cyclosporine, abbreviated as CsA), is a nonpolar cyclic oligopeptide drug composed of 11 amino acid residues. It is soluble in organic solvents but almost insoluble in water.

[0003] Cyclosporine can be used as a local immunomodulator in the eye to treat dry keratitis and conjunctivitis, and to inhibit corneal transplant rejection. Taking advantage of its reduced toxicity, increased ocular surface permeability, and prolonged ocular surface retention time, emulsions have become a novel ocular delivery carrier for cyclosporine. For example, Allergan's Restasis (0.05%) ocular emulsion for treating dry eye is an oil-in-water anionic nanoemulsion; its excipients mainly include glycerin, castor oil, Tween 80, and carbomer. Another example is Santen's 0.1% cyclosporine eye drops (III) (Verkazia), an oil-in-water cationic nanoemulsion used to treat severe vernal keratoconjunctivitis in children and adolescents over 4 years of age; its excipients mainly include medium-chain triglycerides, teroxaprol, cetyldimethylbenzylammonium chloride, glycerin, and poloxamer 188. In particular, Santen's cyclosporine emulsion utilizes cationic emulsion technology to increase the drug's residence time in the eye and its distribution in ocular tissues, resulting in better bioavailability and longer-lasting effects of cyclosporine in the tear film. This provides a new and effective treatment option for patients who are intolerant to or unresponsive to existing therapies.

[0004] In existing technologies, pharmacopoeias of several countries provide analytical methods for related substances in cyclosporine raw materials / preparations. However, research has found that when applying these analytical methods to detect cyclosporine emulsions containing tylosporin and medium-chain triglycerides, such as the commercially available 0.1% cyclosporine eye drops from Santen Pharmaceutical Co., Ltd., the main peak and multiple impurity peaks fail to meet the resolution requirements. For example, the cyclosporine main peak cannot be effectively separated from the pro-cyclosporine U (i.e., resolution < 1.5). Secondly, due to the complexity of the raw materials and matrix of oil-in-water cyclosporine emulsions, it is difficult to distinguish excipients from impurities. Excipients interfere with the detection of impurities, especially the tylosporin and medium-chain triglyceride excipient peaks, which significantly interfere with the separation and detection of the cyclosporine main peak and its impurities U, G, and D, resulting in inaccurate test results and consequently affecting the accuracy of drug quality assessment.

[0005] Medium-chain triglycerides are natural oils extracted from palm oil / coconut oil. Their main components are C8 and C10 medium-chain fatty acids. They exhibit good compatibility with various organic solvents, oils, and fat-soluble vitamins, and are commonly used as the oil phase in emulsions. However, there are few reports on the detection and analysis of medium-chain triglycerides using liquid chromatography.

[0006] Tyloxapol, also known as tetrabutylphenol aldehyde or tetrabutylphenol alcohol, is an alkyl aryl polyether alcohol nonionic liquid polymer. It is viscous and oily, soluble in water, benzene, toluene, and chloroform, and acts as an emulsifier in oil-in-water emulsions, significantly affecting the stability of the emulsion system. In existing technologies, Chinese patent CN116026953A discloses an HPLC method for determining the content of tyloxapol. This method uses a high-performance liquid chromatograph with two size-exclusion columns in series, using a buffer-acetonitrile mobile phase to separate and determine tyloxapol in the sample. Another Chinese patent CN117571642A discloses a method for detecting tyloxapol using a UV-Vis spectrophotometer. Neither of these patents addresses the detection and separation of tyloxapol in emulsions.

[0007] Solving the problem of effective separation of related substances and excipient interference in the detection of cyclosporine emulsions has become a major issue that urgently needs to be addressed. Specifically, for Santen's 0.1% cyclosporine eye drops (III) (Verkazia), standards for the formulation are not currently included in pharmacopoeias and patent literature in various countries. Furthermore, the inventors have found that there are few reported methods for simultaneously detecting multiple (>4) related substances in cyclosporine oil-in-water emulsions containing tylosap and medium-chain triglycerides.

[0008] A search revealed Chinese patent publication CN115266996A, which discloses an analytical method for related substances in cyclosporine A ophthalmic gel formulations. This method uses a phosphoric acid-acetonitrile solution / phosphoric acid-isopropanol solution / phosphoric acid aqueous solution as the mobile phase and investigates how to address interference from excipients such as polyoxyethylene castor oil. It can also separate six impurities (cyclosporine B, cyclosporine C, cyclosporine D, cyclosporine H, ifosporine A, and ifosporine H). However, this patent does not address the detection of cyclosporine U and cyclosporine G, nor does it address interference from excipients such as tylosap and medium-chain triglycerides.

[0009] The literature "Chen Yuxiang et al. Preparation and Content Determination of Cyclosporine Ophthalmic Emulsion. Today's Pharmacy, January 2017, Vol. 27, No. 1" discloses a method for detecting the content of cyclosporine in ophthalmic emulsions, using a Gemini C8 column as the chromatographic column and an acetonitrile-water-methanol-phosphoric acid (550:400:50:0.5) mobile phase. The excipients in the emulsion formulation include castor oil, Tween 80, carbomer 981, and glycerin, but do not contain teloxaprol, poloxamer, or medium-chain triglycerides. This patent does not describe a method for detecting cyclosporine impurities, nor does it provide technical guidance on how to address the interference from excipients such as teloxaprol and medium-chain triglycerides.

[0010] The literature "Pang Wenzhe et al. Determination of cyclosporine content and related substances in capsules and soft capsules by HPLC. Chinese Journal of Antibiotics, June 2016, Vol. 41, No. 6" uses tetrahydrofuran-0.05mol / L phosphoric acid solution (45:55) as the mobile phase to detect cyclosporine and its six impurities (cyclosporine C, cyclosporine B, cyclosporine G, cyclosporine H, ifosporine H, and ifosporine A) in cyclosporine capsule samples. This patent does not address the separation of cyclosporine U from the main peak, nor the detection of cyclosporine D and dihydrocyclosporine A, and it does not provide solutions for interference from excipients such as tylosporin.

[0011] In summary, existing technologies cannot meet the needs of quality monitoring of cyclosporine emulsions, especially those containing excipients such as tylosap and medium-chain triglycerides. Therefore, it is necessary to design a related substances detection method that is highly specific for cyclosporine emulsions containing tylosap and medium-chain triglycerides, can distinguish excipients from impurities, and can detect as many impurities as possible. Summary of the Invention

[0012] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method and application for the separation and determination of cyclosporine and its related substances in oil-in-water emulsions using high-performance liquid chromatography (HPLC). Specifically, this invention provides a method for simultaneously distinguishing and detecting the main cyclosporine component and at least five cyclosporine impurities (impurity U, impurity H, dihydro A, impurity D, and impurity G) in cyclosporine oil-in-water emulsions, thereby improving the detection standards for related substances in cyclosporine oil-in-water emulsions. The method of this invention has high specificity, high sensitivity, good repeatability, and high recovery rate, and can effectively control the content of related substances in cyclosporine oil-in-water emulsions.

[0013] The basic information of the main component of cyclosporine and the impurity compounds of cyclosporine is as follows:

[0014]

[0015]

[0016] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0017] A method for detecting related substances in cyclosporine oil-in-water emulsion, the method comprising the following steps:

[0018] (1) Prepare the sample solution;

[0019] (2) The sample solution was analyzed by high performance liquid chromatography;

[0020] (3) Organize and analyze the results;

[0021] The analytical conditions for the high-performance liquid chromatography method are as follows:

[0022] Chromatographic column: Octadecylsilane-bonded silica gel column;

[0023] Accessories: Stainless steel pipe for front column connection;

[0024] Mobile phase A: Acetonitrile: Aqueous phase: tert-butyl methyl ether: Phosphoric acid = 450-480: 490-540: 25-45: 1, wherein the aqueous phase contains 0.1-0.4% ion-pairing reagent;

[0025] Mobile phase B: Acetonitrile: 0.2% phosphoric acid solution = 70:30–80:20

[0026] Flow rate: 1.0–2.5 mL / min

[0027] Injection volume: 40–60 μL

[0028] Detection wavelength: 205–215 nm

[0029] Column temperature: 70~80℃

[0030] Elution method: gradient elution.

[0031] In the detection method of cyclosporine and its related substances in water-in-oil emulsions described in this invention, the ratio of acetonitrile, aqueous phase, tert-butyl methyl ether and phosphoric acid in the mobile phase A, gradient elution, column temperature and chromatographic column used in high performance liquid chromatography are important factors affecting the detection effect.

[0032] When using mobile phases such as acetonitrile-water-tert-butyl methyl ether and phosphoric acid, or tetrahydrofuran-phosphoric acid systems reported in existing pharmacopoeias of various countries, for example, referring to the detection method for cyclosporine raw materials in the Chinese Pharmacopoeia 2020, it was found that the separation between cyclosporine U and the main peak was poor, with a resolution <1.5. Even by adjusting the column temperature and the proportions of each mobile phase, it was difficult to achieve good separation of the main peak and multiple impurities simultaneously, especially the influence of excipient peaks such as tylosap and medium-chain triglycerides. The inventors of this invention accidentally discovered that adding ion-pairing reagents within a specific range to the aqueous mobile phase (A) not only increased the overall retention time of the components and impurity peaks but also improved the resolution between the main peak and pre-heterocyclosporine U, while ensuring that excipient peaks did not interfere with the detection.

[0033] Specifically, the ion-pairing reagent added to the aqueous phase of mobile phase A has a content of 0.1% to 0.4%. A content below 0.1% cannot effectively separate the baselines of each impurity, while a content above 0.4% results in delayed peak elution and interference from excipient peaks. Furthermore, excessively high concentrations of ion-pairing reagent can affect column life. Preferably, the ion-pairing reagent content in the aqueous phase is 0.2%. On the other hand, the choice of ion-pairing reagent has varying effects on the retention times of the main peak and each impurity peak. Through multiple experiments, it was found that the ion-pairing reagent added to the aqueous phase of mobile phase A can be tetrabutylammonium hydroxide or dodecyltrimethylammonium chloride. Considering factors such as baseline, elution time, peak shape, and resolution, the most preferred ion-pairing reagent is tetrabutylammonium hydroxide.

[0034] Furthermore, the ratio of acetonitrile, aqueous phase, and tert-butyl methyl ether in mobile phase A used in the high-performance liquid chromatography (HPLC) method all affect the detection results, including peak position, separation of the main peak and impurities, and the appearance of excipient peaks. The inventors discovered that reducing the proportion of tert-butyl methyl ether shifts the peak position later, reducing the influence of excipient peaks; increasing the proportion of acetonitrile advances the peak position, but reduces the separation of the main peak and impurity peaks, resulting in poorer peak shape. To balance detection results and eliminate the influence of excipients, the ratio of acetonitrile, aqueous phase, and tert-butyl methyl ether in mobile phase A needs to be within a specific range. After numerous experiments, the inventors creatively discovered that when mobile phase A is a mixed solution of acetonitrile: aqueous phase (containing 0.1–0.4% ion-pairing reagent): tert-butyl methyl ether: phosphoric acid = 450–480: 490–540: 25–45: 1 (volume ratio), effective separation and detection of cyclosporine and its five impurities can be achieved. To ensure the achievement of the objectives of this discovery, preferably, the mobile phase A is acetonitrile: aqueous phase (containing 0.2% ion-pairing reagent): tert-butyl methyl ether: phosphoric acid = 460-470: 500-515: 30-40: 1; more preferably, the mobile phase A is acetonitrile: aqueous phase (containing 0.2% ion-pairing reagent): tert-butyl methyl ether: phosphoric acid = 460-470: 500-510: 30-38: 1; specifically, the mobile phase A can be acetonitrile: aqueous phase (containing 0.2% ion-pairing reagent): tert-butyl methyl ether: phosphoric acid = 465: 502: 33: 1.

[0035] The B phase of the mobile phase in this invention is a solution obtained by mixing acetonitrile and 0.2% phosphoric acid water. In this discovery, mobile phase B ensures effective separation of various impurity peaks, and on the other hand, ensures effective elution of excipient components after the impurity peaks have eluted. Specifically, the inventors have found that a single acetonitrile / methanol / tert-butyl methyl ether solvent is insufficient to simultaneously achieve the effects of this invention; only when acetonitrile and phosphoric acid water are used in appropriate proportions can the purpose of this invention be achieved; preferably, the mobile phase B of this invention is a solution obtained by mixing acetonitrile:0.2% phosphoric acid water in a volume ratio of 70:30 to 80:20; most preferably, the mobile phase B of this invention is a solution obtained by mixing acetonitrile:0.2% phosphoric acid water in a volume ratio of 75:25.

[0036] In the detection method for cyclosporine and related substances in its oil-in-water emulsions according to the present invention, a gradient elution method is employed. The setting of the elution gradient is a crucial technical factor in achieving effective detection. Specifically, researchers have found that, using the mobile phase of the present invention, the elution time of the main peak is between 40 and 50 minutes, while the elution times of impurities D and G are between 50 and 70 minutes. However, the peaks of tylosporin and medium-chain triglyceride excipients in the emulsion emulsion are around 30-45 minutes and 50-70 minutes, respectively. This results in overlapping and / or coating and / or poor peak shape among impurity peaks when detecting the emulsion, hindering baseline separation of the main peak and impairing the qualitative and quantitative detection of impurities (such as impurities G and D). To ensure the peak shape and separation of the relevant elution peaks, the gradient elution is set as follows:

[0037]

[0038] A preferred gradient setting for this invention is as follows:

[0039]

[0040] A preferred gradient setting for this invention is as follows:

[0041]

[0042] A preferred gradient setting for this invention is as follows:

[0043]

[0044] In this technical solution, the selection of the chromatographic column packing material, manufacturer, model, and specifications has a significant impact on the impurity separation and detection effects. After screening various chromatographic columns, technicians found that only by selecting C18 bonded pentafluorophenyl silica gel packing material, C18 bonded amide silica gel packing material, and C18 silica gel columns from specific manufacturers can the expected detection effects of this invention be achieved. Furthermore, for this invention, the corresponding chromatographic columns are ACE Excel 5C18-PFP (C18 bonded pentafluorophenyl silica gel), ACE Excel 3C18-Amide (C18 bonded amide silica gel), and Agilent Poroshell 120SB-C18 chromatographic columns; further considering factors such as column performance and separation effect, the optimal chromatographic column and specification is ACE Excel·5C18-PFP (4.6mm×250mm, 5μm), which provides the best impurity separation and detection effects.

[0045] In this technical solution, to ensure the accuracy and repeatability of the test results, a stainless steel tube needs to be connected in front of the column, and the stainless steel tube must maintain the same temperature as the column. Furthermore, the stainless steel tube can be Φ0.3mm×1m or Φ0.25mm×2m; the size of the tube has no significant impact on the test results.

[0046] Column temperature is also a crucial factor determining separation efficiency. Specifically, the separation degree between impurity peaks and the main peak gradually increases with increasing column temperature. During development, engineers found that, based on existing pharmacopoeia guidelines, the column temperature needs to be maintained at at least 80°C to ensure effective separation of the cyclosporine main component from the adjacent cyclosporine impurity U. However, excessively high column temperatures cause significant wear and tear on the instrument and column, and may lead to further degradation of cyclosporine or its impurities. In the technical solution of this application, based on the chromatographic column and mobile phase system described herein, a column temperature of 70–75°C is sufficient. Considering factors such as column pressure and elution time, a column temperature of 75°C provides the best separation effect.

[0047] In this technical solution, the flow rate of the mobile phase is preferably controlled between 1.8 and 2.2 mL / min. Specifically, the flow rate control should take into account both detection efficiency and resolution. If the flow rate of the mobile phase is too high, it will accelerate the elution rate and shorten the peak elution time, which is not conducive to achieving the separation effect and will damage the chromatographic column. On the other hand, if the flow rate is too low, it will reduce the elution rate, delay the peak elution time, and also broaden the peak width of each component, which is also not conducive to achieving the separation effect and will correspondingly reduce the detection efficiency. Preferably, the detection effect is best when the flow rate is 2.0 mL / min.

[0048] In this technical solution, to ensure the accuracy of the detection results and the separation effect, the injection volume needs to be controlled at 40-60 μL. Under this condition, cyclosporine and various impurities show a good linear relationship. Preferably, the injection volume is 50 μL, which can maximize the elution effect of the mobile phase, facilitate the separation of various component peaks, and result in the most accurate detection results.

[0049] Based on the publicly available information regarding the detection of cyclosporine-related substances in existing technologies, the preferred detection wavelength in this technical solution is 210 nm.

[0050] In the detection method for cyclosporine and its oil-in-water emulsions described in this invention, the preparation of the test solution can follow the conventional high-performance liquid chromatography (HPLC) method for preparing test sample solutions. Preferably, when the test solution is a 0.1% cyclosporine oil-in-water emulsion, no sample pretreatment is required, and the sample can be directly injected, avoiding the risk of introducing other impurities during pretreatment and simplifying the detection procedure.

[0051] Furthermore, the inventors also accidentally discovered during experiments that, even after the test sample has undergone destructive treatment, the method of this invention is also suitable for detecting newly emerging unknown impurity peaks, particularly hydrolytic impurities of cyclosporine (RRT approximately 1.19), with a resolution >1.5. This indicates that the detection method for cyclosporine emulsions of this invention has broad applicability and can be used to monitor the generation of unknown impurities during the preparation / storage of cyclosporine formulations, ensuring product quality.

[0052] As a preferred embodiment of the present invention, the method for detecting cyclosporine and related substances in its oil-in-water emulsion includes the following steps:

[0053] (1) Prepare the sample solution;

[0054] (2) The sample solution was analyzed by high performance liquid chromatography;

[0055] (3) Organize and analyze the results;

[0056] The analytical conditions for the high-performance liquid chromatography method are as follows:

[0057] Column: ACE Excel 5C18-PFP (4.6mm × 250mm, 5μm)

[0058] Accessories: Stainless steel pipe (Φ0.3mm×1m) for column front connection

[0059] Mobile phase A: Acetonitrile: Aqueous phase: tert-butyl methyl ether: Phosphoric acid = 465:502:33:1, wherein the aqueous phase contains 0.2% ion-pairing reagent;

[0060] Mobile phase B: Acetonitrile: 0.2% phosphoric acid solution = 75:25;

[0061] Detection wavelength: 210nm;

[0062] Column temperature: 75℃;

[0063] Flow rate: 2.0 mL / min;

[0064] Injection volume: 50 μL;

[0065] Perform gradient elution according to the table below:

[0066]

[0067]

[0068] Methodological validation experiments showed that the detection method of this invention can simultaneously detect multiple impurities in cyclosporine, exhibiting high specificity, high precision, and high recovery. Under both undegraded and degraded conditions, the similarity of the main peak's minimum peak purity was ≥ the minimum peak purity threshold. Under each degraded condition, the minimum resolution between the main peak and adjacent impurity peaks was greater than 2. The material balance under each degraded condition ranged from 97.0% to 101.6%, indicating that this method can effectively detect various degradation products. Furthermore, this method demonstrates good durability across different column temperatures (73–77℃), flow rates (1.8–2.2 mL / min), mobile phase ratios, stainless steel tubing of different specifications, and different batches of columns from the same manufacturer.

[0069] The present invention relates to a method for detecting related substances in a cyclosporine oil-in-water emulsion, wherein the cyclosporine oil-in-water emulsion is an ophthalmic emulsion.

[0070] The present invention relates to a method for detecting related substances in a cyclosporine water-in-oil emulsion, wherein the cyclosporine water-in-oil emulsion contains the excipient teroxaprol; specifically, the cyclosporine water-in-oil emulsion further contains the excipient medium-chain triglycerides; more specifically, it also contains hexadecyl dimethyl benzyl ammonium chloride, glycerol, teroxaprol, and poloxamer 188. More specifically, the cyclosporine water-in-oil emulsion is commercially available Santen Corporation's 0.1% cyclosporine eye drops (III) (Verkazia) and its generic counterparts.

[0071] Beneficial effects of this invention:

[0072] 1) This invention uses a chromatographic column packed with C18 bonded pentafluorophenyl silica gel. By optimizing the high performance liquid chromatography conditions, it can ensure that at least the main component of cyclosporine, impurity U, impurity H, impurity dihydrogen A, impurity G and impurity D are all effectively separated in the system suitability solution chromatogram, and the resolution between each peak is greater than 2.0.

[0073] 2) The method of the present invention can separate hydrolyzed impurities that are difficult to separate from the main peak under the same chromatographic conditions, and can effectively detect hydrolyzed impurities in cyclosporine emulsion (RRT about 1.19), and also solves the problem of separation and determination of hydrolyzed impurities in cyclosporine emulsion.

[0074] 3) The test solution of this method does not require pretreatment and can be directly injected for detection, avoiding the residue of emulsions and excipients in the column, reducing the ineffective detection of certain impurities, and thus avoiding the impact on drug research and development, production and medication safety.

[0075] 4) This method is highly specific, accurate, and easy to operate, thus ensuring the safety of cyclosporine emulsions, and providing a technical basis for the quality control of cyclosporine emulsions containing tylosap and medium-chain triglyceride excipients. Attached Figure Description

[0076] Figure 1 Superimposed spectra of solution separation performance for different column temperatures (65℃, 70℃, 75℃, 80℃) to demonstrate the applicability of the system.

[0077] Figure 2 This is the HPLC chromatogram of the system suitability solution obtained in Example 2.

[0078] Figure 3 This is an HPLC overlay of the system suitability solution, untreated and alkali-treated cyclosporine emulsions from Example 3.

[0079] Figure 4 This is a superimposed HPLC spectrum of the system adaptability solution and blank emulsion of Comparative Example 1.

[0080] Figure 5 This is a superimposed HPLC spectrum of the system adaptability solution and blank emulsion of Comparative Example 2.

[0081] Figure 6 The above is a superimposed HPLC spectrum of the system suitability solution and blank emulsion obtained in Comparative Example 3.

[0082] Figure 7 The above is a superimposed HPLC spectrum of the system suitability solution and blank emulsion obtained for Comparative Example 4.

[0083] Figure 8 The above is a superimposed HPLC spectrum of the system suitability solution and blank emulsion obtained in Comparative Example 5. Detailed Implementation

[0084] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the invention are not limited thereto. The cyclosporine emulsion test sample in the following embodiments is derived from commercially available 0.1% cyclosporine eye drops (III) (Verkazia); or a self-developed 0.1% cyclosporine eye drops containing the same excipients as described in patents CN101056615A and CN101534791A; other reagents, unless otherwise stated, are all commercially available analytical grade reagents.

[0085] The preparation methods for the mobile phase solution and sample solutions (including but not limited to test solution, reference solution, system suitability solution, positioning solution, sensitivity solution, etc.) of this invention can be obtained by referring to conventional methods skilled in the art. In particular, for the test sample of this invention, which is cyclosporine emulsion, no treatment is required, and it can be directly injected. As an example, the sample solution preparation process in this invention is as follows:

[0086] Blank emulsion (self-developed): Take the blank emulsion to obtain the blank emulsion.

[0087] Test solution: Take cyclosporine emulsion and inject it directly.

[0088] Reference solution: Weigh an appropriate amount of cyclosporine reference standard accurately, dissolve it in 50% acetonitrile solution and dilute quantitatively to prepare a solution containing approximately 10 μg of cyclosporine per ml.

[0089] Cyclosporine System Suitability Solution (National Institutes for Food and Drug Control): Accurately weigh approximately 10 mg of the cyclosporine system suitability control (National Institutes for Food and Drug Control) and place it in a 10 ml volumetric flask. Add diluent to dissolve and dilute to the final volume, then shake well. The system suitability control is sourced from the National Institutes for Food and Drug Control (containing the main cyclosporine component, impurity U, impurity H, impurity dihydrogen A, impurity G, and impurity D, etc., batch number 130584-201001).

[0090] Example 1 investigates the effect of column temperature on system suitability and solution separation.

[0091] The specific experimental methods and steps are as follows:

[0092] (1) Prepare reference solution, system suitability solution and blank emulsion sample solution;

[0093] (2) The sample solution was analyzed by high performance liquid chromatography;

[0094] (3) Organize and analyze the results;

[0095] The analytical conditions for the high-performance liquid chromatography method are as follows:

[0096] Column: Agilent ZORBAX SB-C18 (4.6×250mm, 5μm)

[0097] Accessories: Stainless steel pipe (φ0.3mm×1m) for column front connection

[0098] Mobile phase A: Acetonitrile: Aqueous phase: tert-butyl methyl ether: Phosphoric acid (430:520:50:1); The aqueous phase does not contain ion-pairing reagents;

[0099] Mobile phase B: Acetonitrile: 0.2% phosphoric acid solution = 75:25

[0100] Detection wavelength: 210nm

[0101] Flow rate: 2.0 mL / min

[0102] Injection volume: 50 μL

[0103] Perform gradient elution according to the table below:

[0104]

[0105] The effect of different column temperatures on the system's suitability and solution separation performance is shown in Table 1.

[0106] Table 1. Effect of column temperature on system suitability and solution separation.

[0107] Column temperature Separation of cyclosporine and prohexacyclosporine U 65℃ They overlap and cannot be separated. 70℃ They overlap and cannot be separated. 75℃ The package exhibits poor separation, with a resolution of 1.346. 80℃ It meets the requirements, with a resolution of 1.869.

[0108] For detailed HPLC overlay chromatograms of system suitability solutions obtained at various column temperatures, please refer to [link / reference]. Figure 1 Based on the data in Table 1, it can be observed that as the column temperature increases, the separation degree between the cyclosporine main component and prohexacyclosporine U in the system suitability solution increases. When the column temperature is below 75℃, impurity U cannot be effectively separated and detected. Although the separation degree increases to 1.869 at a column temperature of 80℃, and impurity U and the main peak meet the baseline separation requirements, experiments show that under the mobile phase conditions of Example 1, excipient peaks are present at 15–26 min and 45–70 min, affecting the detection of impurity U; furthermore, the relatively high column temperature of 80℃ increases column wear. Therefore, to ensure method specificity and detection effectiveness, further optimization of chromatographic conditions is needed.

[0109] Example 2

[0110] Based on the results of Example 1, and taking into account the impact of temperature on the wear and tear of the chromatographic column and liquid chromatograph, as well as the effect on sample stability, a column temperature of 75℃ was selected for further development. Tetrabutylammonium hydroxide ion-pairing reagent was added to the aqueous phase of mobile phase A, and the method was optimized in terms of the ratio of mobile phase A to mobile phase B, the elution gradient, and the chromatographic column, resulting in the following preferred scheme of this invention:

[0111] The specific experimental methods and steps are as follows:

[0112] (1) Prepare reference solution, system suitability solution and blank emulsion sample solution;

[0113] (2) The sample solution was analyzed by high performance liquid chromatography;

[0114] (3) Organize and analyze the results;

[0115] The analytical conditions for the high-performance liquid chromatography method are as follows:

[0116] Column: ACE Excel 5C18-PFP (4.6mm × 250mm, 5μm)

[0117] Accessories: Stainless steel pipe (φ0.3mm×1m) for column front connection

[0118] Mobile phase A: Acetonitrile: Aqueous phase: tert-butyl methyl ether: Phosphoric acid = 465:502:33:1, the aqueous phase contains 0.2% tetrabutylammonium hydroxide ion-pairing reagent;

[0119] Mobile phase B: Acetonitrile: 0.2% phosphoric acid solution = 75:25

[0120] Detection wavelength: 210nm

[0121] Column temperature: 75℃

[0122] Flow rate: 2.0 mL / min

[0123] Injection volume: 50 μL

[0124] Perform gradient elution according to the table below:

[0125]

[0126]

[0127] The peak results are shown in Table 2 below:

[0128] Table 2. Peak Table for System Suitability Solution Detection in Example 2

[0129]

[0130] As shown in Table 2 above, the technical solution obtained by this invention can effectively distinguish cyclosporine and its five cyclosporine impurities (impurity U, impurity H, impurity dihydro A, impurity D, and impurity G). Furthermore, cyclosporine and each impurity achieve baseline separation with stable baselines and good peak shapes. The excipient peaks do not interfere with the separation of the main peak and impurities, and the resolution of adjacent chromatographic peaks is greater than 2, which is more conducive to identification and localization. The HPLC chromatogram of the system suitability solution in Example 2 is shown below. Figure 2 As shown.

[0131] Example 3

[0132] The detection method of Example 2 was used to test cyclosporine emulsions (self-developed generic drug of commercially available 0.1% cyclosporine eye drops (III): batch ZS01210504) that were neither treated with alkali as the test solution. The results are shown in Table 3 below. The alkali treatment procedure was as follows: 5 ml of the test solution was accurately transferred to a 10 ml volumetric flask, 1 ml of 1 mol / L sodium hydroxide solution was added, and the solution was incubated at room temperature for 5 hours. Then, 1 ml of 1 mol / L hydrochloric acid solution was added to neutralize the solution, and the mixture was diluted to the mark with acetonitrile and shaken well.

[0133] Table 3. Detection results of cyclosporine emulsions that were neither treated with alkali nor subjected to alkali-induced degradation.

[0134]

[0135]

[0136] As shown in Table 3 above and the obtained HPLC chromatograms, this method is applicable not only to the detection of cyclosporine emulsions but also to the detection of cyclosporine emulsions after alkali degradation. In particular, it can detect hydrolysis impurity peaks with an RRT of approximately 1.19, which is beneficial for monitoring the safety of cyclosporine emulsions. The HPLC overlay chromatograms of the system suitability solution, untreated cyclosporine emulsion, and alkali-treated cyclosporine emulsions in Example 3 are shown below. Figure 3 As shown.

[0137] Example 4 Sample Detection

[0138] Using the detection method of Example 2, commercially available 0.1% cyclosporine eye drops (III) were tested as the test solution. The results are shown in Table 4 below. It can be seen that the method of the present invention can accurately distinguish and detect the contents of impurities H, G, U, dihydrogen A and D in cyclosporine.

[0139] Table 4. Results of related substances in cyclosporine emulsion samples.

[0140]

[0141] Comparative Example 1

[0142] The same chromatographic conditions and column as in Example 2 were used. The only difference in the chromatographic conditions was that the elution gradient was not within the scope of protection claimed in this invention. Gradient elution was performed according to the table below:

[0143]

[0144] The superimposed HPLC spectra of the system adaptability and blank emulsion obtained in Comparative Example 1 are shown below. Figure 4 As shown, although cyclosporine and adjacent impurity U can be effectively separated in the spectrum, excipient peaks still exist in the blank emulsion at 60-70 min, interfering with the detection of impurity D and impurity G peaks.

[0145] Comparative Example 2

[0146] The same chromatographic conditions as in Example 2 were used, except that mobile phase A did not contain ion-pairing reagents and mobile phase B was acetonitrile.

[0147] The superimposed HPLC spectra of the system adaptability and blank emulsion obtained from Comparative Example 2 are shown below. Figure 5 As shown, although cyclosporine and the adjacent impurity U can be effectively separated, the excipient peak in the blank emulsion at 55–70 min interferes with the detection of impurity D. Furthermore, the baseline stability of the blank emulsion chromatogram produced by this mobile phase system is relatively poor, with baseline drift occurring at 30–40 min. Therefore, it can be concluded that the technical solutions of not adding ion-pairing reagents to mobile phase A and using acetonitrile as the sole mobile phase B cannot eliminate the interference from excipient peaks.

[0148] Comparative Example 3

[0149] The same chromatographic conditions as in Example 2 were used, except that the solvent ratio in mobile phase A was not within the scope of protection claimed in this invention. Compared with the mobile phase of cyclosporine raw material in the Chinese Pharmacopoeia 2020 (i.e., mobile phase A in Example 1 was acetonitrile: aqueous phase: tert-butyl methyl ether: phosphoric acid = 430:520:50:1), the ratio of aqueous phase was not changed, but the ratio of acetonitrile and tert-butyl methyl ether was adjusted. Specifically, mobile phase A was acetonitrile: aqueous phase: tert-butyl methyl ether: phosphoric acid = 440:520:40:1, and the aqueous phase contained 0.2% tetrabutylammonium hydroxide ion-pairing reagent.

[0150] The HPLC superimposed spectra of the system suitability solution and blank emulsion of Comparative Example 3 are shown below. Figure 6 As shown. Compared with the existing known mobile phase of acetonitrile:water:tert-butyl methyl ether:phosphoric acid = 430:520:50:1, without changing the water phase ratio, by reducing the tert-butyl methyl ether to about 4.0% and increasing the acetonitrile ratio to 44.0%, the peak positions of the main component, impurities, and excipients are all delayed. However, in this mobile phase system, at 60–70 min, there is an excipient peak that interferes with the detection of impurity G.

[0151] Comparative Example 4

[0152] The same chromatographic conditions and column as in Example 2 were used. The only difference in the chromatographic conditions was that the solvent ratio in mobile phase A was not within the scope of protection claimed in this invention. Compared with the mobile phase of cyclosporine raw material in the Chinese Pharmacopoeia 2020 (i.e., mobile phase A in Example 1 was acetonitrile: aqueous phase: tert-butyl methyl ether: phosphoric acid = 430:520:50:1), the acetonitrile ratio was not changed, but the ratio of aqueous phase and tert-butyl methyl ether was adjusted. Specifically, mobile phase A was acetonitrile: aqueous phase: tert-butyl methyl ether: phosphoric acid = 430:530:40:1; the aqueous phase contained 0.2% tetrabutylammonium hydroxide ion-pairing reagent.

[0153] The HPLC superimposed spectra of the system suitability solution and blank emulsion of Comparative Example 4 are shown below. Figure 7 As shown, compared with the existing known mobile phase of acetonitrile:water:tert-butyl methyl ether:phosphoric acid = 430:520:50:1, without changing the acetonitrile ratio, by adjusting the water phase ratio to 53.0% and the tert-butyl methyl ether ratio to 4.0%, the mobile phase system still has an excipient peak interfering with the detection of impurity D at about 70 min.

[0154] Comparative Example 5

[0155] The analysis of related substances in sterile cyclosporine solutions was performed in accordance with the chromatographic methods specified in the European Pharmacopoeia (BP2023). The specific experimental methods and procedures are as follows:

[0156] (1) Prepare reference solution, system suitability solution and blank emulsion sample solution;

[0157] (2) The sample solution was analyzed by high performance liquid chromatography;

[0158] (3) Organize and analyze the results;

[0159] The analytical conditions for the high-performance liquid chromatography method are as follows:

[0160] Column: Agilent ZORBAX SB-C18 (4.6×250mm, 5μm)

[0161] Accessories: Stainless steel pipe (φ0.25mm×2m) for column front connection

[0162] Mobile phase A: Acetonitrile: Aqueous phase: tert-butyl methyl ether: Phosphoric acid = 460:490:50:1, the aqueous phase does not contain ion-pairing reagents;

[0163] Mobile phase B: Acetonitrile

[0164] Detection wavelength: 210nm

[0165] Column temperature: 80℃

[0166] Flow rate: 2.0 mL / min

[0167] Injection volume: 50 μL

[0168] Perform gradient elution according to the table below:

[0169]

[0170] The HPLC superimposed spectra of the system suitability solution and blank emulsion of Comparative Example 5 are shown below. Figure 8 As shown, under these mobile phase conditions, the peak positions are advanced, and the leading peaks accumulate. The main cyclosporine peak elutes between 18 and 20 min, while the excipient peak in the blank emulsion elutes between 15 and 20 min. This prevents effective baseline separation between the main peak and impurity U, and the separation of impurities G and D at the downstream end is also poor. Therefore, these chromatographic conditions are not suitable for the analysis of the cyclosporine emulsion described in this invention.

[0171] In summary, the methods disclosed in the prior art are insufficient to simultaneously separate all impurities in cyclosporine water-in-oil emulsions. Only when the chromatographic conditions, including the elution solvent system and elution gradient, and the chromatographic column, all fall within the protection scope of this invention, can the high-performance liquid chromatography (HPLC) method for detecting cyclosporine water-in-oil emulsions simultaneously separate and detect the main cyclosporine component, impurity U, impurity H, impurity dihydro A, impurity G, and impurity D. Therefore, the analytical method for cyclosporine water-in-oil emulsions described in this invention can be used for monitoring the production process of cyclosporine water-in-oil emulsions and for quality control of the formulation products.

[0172] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting related substances in cyclosporine oil-in-water emulsion, the method comprising the following steps: (1) Prepare the sample solution; (2) The sample solution was analyzed by high performance liquid chromatography; (3) Organize and analyze the results; The analytical conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: Octadecylsilane-bonded silica gel column; Accessories: Stainless steel pipe for front column connection; Mobile phase A: Acetonitrile: Aqueous phase: tert-butyl methyl ether: Phosphoric acid = 450-480: 490-540: 25-45: 1, wherein the aqueous phase contains 0.1%-0.4% ion-pairing reagent; Mobile phase B: Acetonitrile: 0.2% phosphoric acid solution = 70:30 to 80:20; Flow rate: 1.0–2.5 mL / min Injection volume: 40–60 μL Detection wavelength: 205–215 nm Column temperature: 70~80℃ Perform gradient elution using the following elution settings:

2. The detection method according to claim 1, wherein the gradient elution is set as follows:

3. The detection method according to claim 2, wherein the gradient elution setting is preferably as follows:

4. The detection method according to claim 1, wherein the mobile phase A is preferably acetonitrile: aqueous phase: tert-butyl methyl ether: phosphoric acid = 460-470: 500-515: 30-40: 1, wherein the aqueous phase contains 0.2% ion-pairing reagent.

5. The detection method according to claim 1, wherein the ion-pairing reagent is selected from tetrabutylammonium hydroxide and dodecyltrimethylammonium chloride.

6. The detection method according to claim 1, wherein the mobile phase B is a mixed solution of acetonitrile: 0.2% phosphoric acid water = 75:

25.

7. The detection method according to claim 1, wherein the chromatographic column is selected from C18 bonded pentafluorophenyl silica gel packed column, C18 bonded amide silica gel packed column and C18 silica gel packed column from a specific manufacturer; more preferably ACE Excel5C18-PFP, ACE Excel 3C18-Amide and Agilent Poroshell 120SB-C18.

8. According to the detection method of claim 1, the specifications of the stainless steel pipe connecting the column front are Φ0.3mm×1m or Φ0.25mm×2m.

9. The detection method according to claim 1, wherein the cyclosporine oil-in-water emulsion contains the excipients tylosap and medium-chain triglycerides.

10. A method for detecting cyclosporine and related substances in its oil-in-water emulsion, the method comprising the following steps: (1) Prepare the sample solution; (2) The sample solution was analyzed by high performance liquid chromatography; (3) Organize and analyze the results; The analytical conditions for the high-performance liquid chromatography method are as follows: Column: ACE Excel 5C18-PFP (4.6mm × 250mm, 5μm) Accessories: Stainless steel pipe (φ0.3mm×1m) for column front connection Mobile phase A: Acetonitrile: Aqueous phase: tert-butyl methyl ether: Phosphoric acid = 465:502:33:1, wherein the aqueous phase contains 0.2% ion-pairing reagent; Mobile phase B: Acetonitrile: 0.2% phosphoric acid solution = 75:25 Detection wavelength: 210nm Column temperature: 75℃ Flow rate: 2.0 mL / min Injection volume: 50 μL Perform gradient elution according to the table below:

11. The method for detecting related substances in cyclosporine water-in-oil emulsion according to any one of claims 1-10, for use in detecting cyclosporine ophthalmic preparations containing teroxaprol and medium-chain triglycerides.

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