Method for detecting related substances in cyclosporine product and use thereof

Abstract

A method for detecting related substances in a cyclosporine product and the use thereof. The detection method is performed using high-performance liquid chromatography, wherein octadecylsilane bonded silica gel is used as a filler, an aqueous phosphoric acid solution is used as mobile phase A, and tetrahydrofuran is used as mobile phase B to perform gradient elution. By means of screening and optimizing the chromatographic conditions, in particular screening and optimizing the gradient elution program, the method can eliminate interference from excipient peaks in the detection of related substances in cyclosporine, has a high specificity, and can accurately and reliably detect multiple known impurities in an cyclosporine ophthalmic gel, thereby guaranteeing a controlled product quality.

Description

Method for detecting related substances in cyclosporine product and application thereof TECHNICAL FIELD

[0001] The technical scheme of the present application belongs to the field of pharmaceutical preparation analysis, and specifically relates to a method for detecting related substances in a cyclosporine product and application thereof. BACKGROUND

[0002] Cyclosporine (CyA) is a cyclic polypeptide composed of 11 amino acids, which is an active metabolite of a fungus in the soil. Cyclosporine, as an immunosuppressant, is a commonly used anti-inflammatory drug in the field of ophthalmology. Cyclosporine eye gel, as a local immunomodulator, can be used to treat ocular inflammation caused by keratoconjunctivitis sicca and to inhibit the condition in which the patient's tear secretion is inhibited. At present, the quality standards of cyclosporine and some of its preparations have been included in many pharmacopoeias. However, there are differences in the naming and control strategies of impurities in these pharmacopoeias. Therefore, it is crucial to develop a corresponding and effective impurity control strategy and specific analysis method for cyclosporine eye gel, a new type of preparation. These measures are of great significance to ensure the drug quality and safety of cyclosporine eye gel.

[0003] Since cyclosporine is almost insoluble in water, in order to improve its solubility, stability and bioavailability in eye preparations, it needs to be dissolved in lipophilic solvents. For example, cyclosporine eye drops produced by the American ALLERGAN company (Specification: 0.05%) is an oil-in-water cyclosporine emulsion that uses polyoxyethylene castor oil to solubilize. However, polyoxyethylene castor oil is a mixture obtained by reacting glycerol ricinoleate with ethylene oxide at a fixed ratio, which has strong ultraviolet absorption and contains multiple excipient peaks. When using the existing related substance detection method, these excipient peaks will interfere with the determination of impurity content.

[0004] In addition, with the continuous improvement of research on cyclosporine impurities and the increasing demand for drug quality and safety, some new cyclosporine impurities, such as cyclosporine U and dihydrocyclosporine A, have been included in the research scope of related substances of cyclosporine eye preparations. However, the existing method cannot effectively separate cyclosporine U and dihydrocyclosporine A while eliminating the interference of excipients. SUMMARY

[0005] The purpose of the present application is to effectively separate and detect cyclosporine U and dihydrocyclosporine A in cyclosporine related products using high performance liquid chromatography, and to avoid the interference of excipients, thereby providing a method for detecting related substances in a cyclosporine product and application thereof.

[0006] To achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is as follows:

[0007] In a first aspect, the application provides a method for detecting related substances in a cyclosporin product, which is detected by high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography comprise:

[0008] The chromatographic conditions of the high performance liquid chromatography comprise:

[0009] 0-8 min, the volume percentage of the mobile phase A is 68%; 8-75 min, the volume percentage of the mobile phase A is 68%→67%; 75-135 min, the volume percentage of the mobile phase A is 67%→65%; 135-135.01 min, the volume percentage of the mobile phase A is 65%→20%; 135.01-140 min, the volume percentage of the mobile phase A is 20%; 140-140.01 min, the volume percentage of the mobile phase A is 20%→68%; 140.01-150 min, the volume percentage of the mobile phase A is 68%.

[0010] In an optional embodiment, the chromatographic conditions of the high performance liquid chromatography further comprise at least one of the following:

[0011] (1) the chromatographic column is Agilent AdvanceBio Peptide Map, 150 mm×4.6 mm, 2.7 μm;

[0012] (2) the concentration of phosphoric acid in the aqueous phosphoric acid solution is 0.05%-0.2%;

[0013] (3) the flow rate is 0.9-1.2 mL / min;

[0014] (4) the column temperature is 58-60°C;

[0015] (5) the injection volume is 50-100 μl;

[0016] (6) the detection wavelength is 216-220 nm.

[0017] In an optional embodiment, the method further comprises a step of preparing a test sample solution by using a test sample, wherein the test sample is added with a diluent, dissolved, added with a calcium chloride solution, solid-liquid separated, and the liquid is taken;

[0018] Optionally, the diluent is a mixed solution of tetrahydrofuran and water, and the volume ratio of tetrahydrofuran to water is (3-4):(7-6);

[0019] Optionally, the content of calcium chloride in the calcium chloride solution is 5%-20%;

[0020] Optionally, the diluent is used in an amount of 8-10 mL and the calcium chloride solution is used in an amount of 0.1-0.5 mL relative to 2 mg of cyclosporine.

[0021] Optionally, the solid-liquid separation is centrifugation at a speed of 8000-12000 rpm for 5-20 min.

[0022] In an optional embodiment, the detection method further comprises the step of separately preparing a control solution by separately dissolving a control substance in a diluent.

[0023] The control substance comprises cyclosporine control substance, cyclosporine B control substance, cyclosporine C control substance, cyclosporine D control substance, cyclosporine H control substance, cyclosporine U control substance, dihydro-cyclosporine A control substance, isocyclosporine A control substance and isocyclosporine H control substance.

[0024] In an optional embodiment, the concentration of the cyclosporine control substance is 2 μg / mL, the concentration of the cyclosporine B control substance is 2 μg / mL, the concentration of the cyclosporine C control substance is 2 μg / mL, the concentration of the cyclosporine D control substance is 2 μg / mL, the concentration of the cyclosporine H control substance is 2 μg / mL, the concentration of the cyclosporine U control substance is 2 μg / mL, the concentration of the dihydro-cyclosporine A control substance is 2 μg / mL, the concentration of the isocyclosporine A control substance is 2 μg / mL and the concentration of the isocyclosporine H control substance is 2 μg / mL in the respective control solution.

[0025] In an optional embodiment, the content of each related substance is calculated by the principal component external standard method with a correction factor. Specifically, the content of each related substance can be calculated according to the following formula:

[0026] In the above formula, Cs is the concentration of the cyclosporine control substance solution; Ms is the weight of the cyclosporine control substance; P is the purity of the cyclosporine control substance; Vs is the dilution volume of the cyclosporine control substance; A is the peak area of any impurity in the test solution; As is the peak area of the cyclosporine in the control solution; V is the dilution volume of the test solution; M is the sample weight; L is the labeled amount of the cyclosporine related product; and f is the correction factor corresponding to each related substance (impurity).

[0027] The correction factor corresponding to isocyclosporine H is 1.44, the correction factor corresponding to isocyclosporine A is 1.19, the correction factor corresponding to cyclosporine C is 1.00, the correction factor corresponding to cyclosporine B is 1.32, the correction factor corresponding to cyclosporine U is 1.14, the correction factor corresponding to dihydro-cyclosporine A is 1.00, the correction factor corresponding to cyclosporine D is 1.13, the correction factor corresponding to cyclosporine H is 1.00 and the correction factor corresponding to unknown impurity is 1.00.

[0028] In an alternative embodiment, the cyclosporin product comprises a cyclosporin raw material and a cyclosporin formulation;

[0029] Optionally, the cyclosporin formulation comprises a formulation with polyoxyethylene castor oil as an excipient.

[0030] In an alternative embodiment, the cyclosporin formulation comprises a cyclosporin gel, and the cyclosporin gel uses polyoxyethylene castor oil as a solubilizer.

[0031] In an alternative embodiment, the cyclosporin gel comprises the following components:

[0032] Cyclosporin 0.05% to 0.1% (w / w), polyoxyethylene castor oil 0.95% to 1.05% (w / w), and gel base 0.04% to 0.10% (w / w). The gel base can be selected within a certain range, for example, the gel base can be carbomer.

[0033] In a second aspect, the application also provides the use of the above-mentioned detection method in the quality control of cyclosporin products.

[0034] Based on the above technical solutions, the application has at least the following beneficial effects:

[0035] (1) The detection method provided by the application uses high performance liquid chromatography for detection, with octadecylsilane-bonded silica gel as the filler, and aqueous phosphoric acid as the mobile phase A and tetrahydrofuran as the mobile phase B for gradient elution. This detection method optimizes the chromatographic conditions by screening, especially the gradient elution program, which can exclude the interference of excipient peaks on the detection of cyclosporin related substances, has strong specificity, and can accurately and reliably detect multiple known impurities in cyclosporin eye gel, thereby ensuring controllable product quality.

[0036] Specifically, the detection method can effectively separate and accurately detect two impurities, cyclosporin U and dihydrocyclosporin A, while eliminating the interference of polyoxyethylene castor oil excipient peaks and ensuring the normal separation and detection of the other six known impurities (cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin H, isocyclosporin A, and isocyclosporin H).

[0037] (2) The detection method provided by the application has stronger impurity detection capability without excipient interference, and can effectively separate and detect cyclosporin U and dihydrocyclosporin A in cyclosporin eye gel. After multiple batches of continuous sample injection, the retention time of the chromatographic peak is stable, avoiding the problem of overlapping impurity positioning and inaccurate content determination caused by chromatographic peak drift. This detection method provides a solid and effective foundation for impurity research and control strategy of cyclosporin related products (such as eye gel), thereby ensuring drug quality and safety. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the specific embodiments or prior art of the present application, the drawings required to be used in the description of the specific embodiments or prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0039] Figure 1 is a chromatogram obtained in Example 1 of the present application;

[0040] Figure 2 is a chromatogram obtained in Test (1) of Example 2 of the present application;

[0041] Figure 3 is a chromatogram obtained in Test (2) of Example 2 of the present application;

[0042] Figure 4 is a chromatogram obtained in Test (3) of Example 2 of the present application;

[0043] Figure 5 is a chromatogram obtained in Test (4) of Example 2 of the present application. DETAILED DESCRIPTION

[0044] The following examples are provided to better further understand the present application and are not limited to the best mode, and do not limit the content and protection scope of the present application. Any person under the inspiration of the present application or the combination of the present application with other prior art features can obtain any product same or similar to the present application, which falls within the protection scope of the present application. If the specific experimental steps or conditions are not indicated in the examples, the operation or conditions can be carried out according to the conventional experimental steps described in the literature in the art. If the reagents or instruments are not indicated by the manufacturer, they are conventional reagent products that can be obtained by market purchase.

[0045] The present application will be further described in detail below in combination with specific embodiments, which cannot be understood as limiting the scope of the present application.

[0046] The instruments, reagents and reagents involved in the embodiments of the present application are as follows:

[0047] Instruments: high performance liquid chromatograph (manufacturer: Agilent Technologies, Inc., model: 1260II); chromatographic column (Agilent AdvanceBio Peptide Map, 150mm x 4.6mm, 2.7μm; Waters Nova-Pak C18, 300mm x 3.9mm, 4μm).

[0048] Reagent: Tetrahydrofuran (Manufacturer: TEDIA, Batch number: 23015132); Anhydrous calcium chloride (Manufacturer: Shanghai McLean Biotechnology Co., Ltd., Batch number: C10463871).

[0049] Reagent: Cyclosporine eye gel (Source: Zhejiang Meikailin Biotechnology Co., Ltd., Batch number: 20200101, 20230101, 20240101; The product contains 0.05% cyclosporine, and other excipients are polyoxyethylene castor oil, propylene glycol, etc.);

[0050] Cyclosporine raw material (Source: Zhejiang Ruibang Pharmaceutical Co., Ltd., Batch number: 10-22101001);

[0051] Cyclosporine reference substance (Source: China Institute for Drug Control, Number: 130495, Batch number: 130495-201904);

[0052] Cyclosporine B reference substance (Source: CATO Research Chemicals Inc., Catalog number: C4X-19981, Batch number: 1027-RC-0004);

[0053] Cyclosporine C reference substance (Source: CATO Research Chemicals Inc., Catalog number: C4X-19986, Batch number: 0901-RC-0005);

[0054] Cyclosporine D reference substance (Source: CATO Research Chemicals Inc., Catalog number: C4X-19987, Batch number: 1031-RD-0021);

[0055] Cyclosporine H reference substance (Source: CATO Research Chemicals Inc., Catalog number: C4X-19989, Batch number: 0509-RD-0065);

[0056] Cyclosporine U reference substance (Source: CATO Research Chemicals Inc., Catalog number: C4X-19982, Batch number: 1219-RD-0031);

[0057] Dihydrocyclosporine A reference substance (Source: CATO Research Chemicals Inc., Catalog number: C4X-19988, Batch number: 0531-RD-0012);

[0058] Isocyclosporine A reference substance (Source: CATO Research Chemicals Inc., Catalog number: C4X-13111, Batch number: 0325-RD-0017);

[0059] Isocyclosporine H (trifluoroacetate salt) reference substance (source: Toronto Research Chemicals, item number: TRC-I811555, batch number: 8-SKR-30-1, 10-NBP-73-1);

[0060] Cyclosporine system suitability reference substance (source: China Institute for Drug Control, number: 130584, batch number: 130584-202202);

[0061] Blank excipient (source: Zhaoke (Guangzhou) Ophthalmic Pharmaceutical Co., Ltd., batch number: 2022100801, 2023122801; this product is a preparation without cyclosporine, and the excipients and their amounts are consistent with those of cyclosporine eye gel).

[0062] Example 1

[0063] The related substance determination of cyclosporine eye gel (batch number: 20240101) was carried out according to the following method:

[0064] (1) Preparation of diluent: tetrahydrofuran-water (3:7), accurately measure tetrahydrofuran 300 ml and water 700 ml, mix well, and obtain.

[0065] (2) Preparation of 10% CaCl2 solution: weigh anhydrous calcium chloride 5 g, place it in a 50 ml volumetric flask, dissolve with appropriate amount of water, cool to room temperature, dilute to the mark with water, and shake well to obtain.

[0066] (3) Preparation of test solution: take 4 g of the product (equivalent to about 2 mg of cyclosporine), accurately weigh, place in a 10 ml volumetric flask, add appropriate amount of diluent, vortex mix well, ultrasonic (intensity 100, 15 min) to dissolve, cool, add 0.2 ml of 10% calcium chloride solution, dilute to the mark with diluent, shake well, centrifuge (10000 rpm, 10 min), and take the supernatant as the test solution.

[0067] (4) Preparation of reference stock solution: accurately weigh cyclosporine reference substance, cyclosporine B reference substance, cyclosporine C reference substance, cyclosporine D reference substance, cyclosporine H reference substance, cyclosporine U reference substance, dihydrocyclosporine A reference substance, isocyclosporine A reference substance, and isocyclosporine H (trifluoroacetate salt) reference substance, dissolve and dilute quantitatively with tetrahydrofuran to prepare a stock solution containing about 0.2 mg of cyclosporine and 0.1 mg of each impurity reference substance per 1 ml.

[0068] (5) Preparation of positioning solution (control solution, total of 9): precisely pipette cyclosporine control solution 0.1 ml, each impurity control solution 0.2 ml, respectively, into different 10 ml volumetric flasks, dilute to the mark with diluent, shake well, and use as positioning solution (control solution).

[0069] (7) High performance liquid chromatography detection:

[0070] The chromatographic column was Agilent AdvanceBio Peptide Map, 150 mm x 4.6 mm, 2.7 μm; 0.1% phosphoric acid was used as mobile phase A, and tetrahydrofuran was used as mobile phase B, gradient elution was performed according to Table 1; the detection wavelength was 218 nm; the column temperature was 60°C; the flow rate was 1.0 ml / min; and the injection amount was 100 μL.

[0071] Precisely pipette 100 μL of the test solution and each positioning solution, respectively, into a high performance liquid chromatograph to obtain the test results.

[0072] Table 1 Gradient elution table

[0073] The detection results are shown in Figure 1. Figure 1 is a comparison of the chromatogram of the test sample and the chromatogram of each control. The content of each related substance was calculated according to the principal component external standard method with a correction factor, and the calculation formula was:

[0074] In the formula:

[0075] Cs: cyclosporine control solution concentration, mg / ml;

[0076] Ms: cyclosporine control solution weight, mg;

[0077] P: cyclosporine control solution purity, %;

[0078] Vs: cyclosporine control solution dilution volume, ml;

[0079] A: peak area of any impurity in the test solution;

[0080] As: cyclosporine peak area of cyclosporine control solution;

[0081] V: test sample dilution volume, ml;

[0082] M: test sample weight, g;

[0083] L: cyclosporine eye gel labeled amount, 0.5 mg / g;

[0084] f: correction factor.

[0085] The relative retention time and the corresponding correction factor of each impurity relative to cyclosporine (main peak) are shown in Table 2.

[0086] Table 2 Relative retention time and the corresponding correction factor of each impurity

[0087] The peak area of each impurity in the test sample solution, the peak area of cyclosporine in the cyclosporine reference solution, and the calculation results of the content of each impurity are shown in Table 3.

[0088] Table 3 Peak area of impurities, peak area of cyclosporine reference, and calculation results of the content of each impurity

[0089] Example 2

[0090] Determination of related substances of cyclosporine ophthalmic gel (batch number: 20200101, 20231001):

[0091] (1) Initial chromatographic conditions

[0092] Octadecylsilane-bonded silica gel as the filler (Waters Nova-Pak C18, 300 mm x 3.9 mm, 4 μm); the mobile phase is tetrahydrofuran-water-phosphoric acid (400:600:1.58); the detection wavelength is 220 nm; the column temperature is 65°C; the flow rate is 0.8 ml / min; the injection volume is 100 μl. Analyze the peak appearance of each related substance, and the chromatographic results are shown in Figure 2. The results of Figure 2 show that with the increase of the test amount and the test time, the excipient cannot be completely eluted within the running time, and the cyclosporine impurity peaks are interfered by the excipient to different degrees.

[0093] (2) Optimization of the proportion of the mobile phase

[0094] Adjust the proportion of the mobile phase, and the mobile phase is changed to tetrahydrofuran-water-phosphoric acid (380:620:1.2), and other chromatographic conditions are consistent with the initial chromatographic conditions in (1). The chromatographic results are shown in Figure 3. The results of Figure 3 show that the excipient interference is significantly reduced, but with the increase of the test amount and the test time, the cyclosporine impurity peaks appear different degrees of drift, affecting the accuracy of the detection of related substances.

[0095] (3) Adjustment of the chromatographic column

[0096] The problem of peak drift caused by the decrease of column efficiency with the increase of test amount and test time was solved by adjusting the chromatographic column. The adjusted chromatographic column was Agilent AdvanceBio Peptide Map, 150 mm x 4.6 mm, 2.7 μm. To adapt to the chromatographic column, the chromatographic conditions were adjusted as follows: mobile phase A (0.1% phosphoric acid), mobile phase B (tetrahydrofuran); column temperature was 58°C; flow rate was 0.65 ml / min; gradient elution program was as follows: from 0 to 15 min, the volume percentage of mobile phase A was 68%, and the volume percentage of mobile phase B was 32%; from 15 to 80 min, the volume percentage of mobile phase A was 60%, and the volume percentage of mobile phase B was 40%; from 80 to 90 min, the volume percentage of mobile phase A was 40%, and the volume percentage of mobile phase B was 60%; from 90 to 100 min, the volume percentage of mobile phase A was 68%, and the volume percentage of mobile phase B was 32%. The chromatographic results are shown in FIG. 4. After using the above chromatographic column, the retention time of each cyclosporine impurity peak was stable, and did not drift with the increase of test amount and test time, while the interference of excipients was excluded. However, cyclosporine U and dihydrocyclosporine A were eluted at the same retention time, which could not be effectively separated, affecting the detection ability of individual impurities.

[0097] (4) Optimization of gradient elution program

[0098] By optimizing the elution program, cyclosporine U and dihydrocyclosporine A were successfully separated under the premise of avoiding excipient interference and effectively separating other cyclosporine impurities. The specific chromatographic conditions were implemented as follows: detection wavelength was 218 nm; column temperature was 60°C; flow rate was 1.0 ml / min; gradient elution was performed according to Table 4.

[0099] Table 4. Optimized gradient elution program

[0100] The chromatographic results are shown in FIG. 5. As can be seen from FIG. 5, the optimized chromatographic conditions can effectively separate and detect cyclosporine U, dihydrocyclosporine A and other 6 cyclosporine-related impurities, with stable retention time and smooth baseline, and the excipient peaks do not interfere with the detection of impurities.

[0101] Example 3: Specificity experiment

[0102] Diluent: accurately measure 300 ml of tetrahydrofuran and 700 ml of water, mix well, and obtain.

[0103] 10% CaCl2 solution: weigh 5 g of anhydrous calcium chloride, place it in a 50 ml volumetric flask, dissolve with appropriate amount of water, cool to room temperature, dilute to the mark with water, and shake well to obtain.

[0104] Blank adjuvant solution: take the blank adjuvant (not containing cyclosporine) 4g, accurately weigh and place in a 10ml volumetric flask, add the diluent, vortex mix uniformly, then ultrasonic dissolve, cool, add 10% calcium chloride solution 0.2ml, dilute to the mark with diluent, shake well, centrifuge (10000rpm, 10min), take the supernatant as the blank adjuvant solution.

[0105] Test solution: take the product (batch number: 20230101) 4g (about equivalent to 2mg of cyclosporine), accurately weigh and place in a 10ml volumetric flask, add the diluent, vortex mix uniformly, then ultrasonic dissolve (intensity 100, 15min), cool, add 10% calcium chloride solution 0.2ml, dilute to the mark with diluent, shake well, centrifuge (10000rpm, 10min), take the supernatant as the test solution.

[0106] Cyclosporine raw material test solution: accurately weigh cyclosporine raw material, dissolve and dilute to prepare a solution containing about 0.2mg of cyclosporine per 1ml.

[0107] Reference solution: accurately weigh cyclosporine reference, cyclosporine B reference, cyclosporine C reference, cyclosporine D reference, cyclosporine H reference, cyclosporine U reference, dihydrocyclosporine A reference, isocyclosporine A reference, isocyclosporine H (trifluoroacetate) reference, dissolve in tetrahydrofuran and dilute to prepare a reference solution containing about 0.2mg of cyclosporine reference and 0.1mg of each impurity reference per 1ml.

[0108] Cyclosporine reference solution: accurately weigh cyclosporine reference, dissolve and dilute to prepare a solution containing about 2μg of cyclosporine per 1ml.

[0109] Sensitivity solution: accurately measure 1ml of cyclosporine reference solution, place in a 10ml volumetric flask, dilute to the mark with diluent to prepare a solution containing about 0.2μg of cyclosporine per 1ml.

[0110] Positioning solution (total of 9): accurately measure 0.1ml of cyclosporine reference stock solution and 0.2ml of each impurity reference stock solution, place in different 10ml volumetric flasks, dilute to the mark with diluent, shake well, and respectively as positioning solution.

[0111] System suitability solution: accurately weigh 2mg of cyclosporine system suitability reference, accurately measure 0.1ml of cyclosporine H and isocyclosporine H stock solution, place in a 10ml volumetric flask, dissolve and dilute to prepare a solution containing about 0.2mg of cyclosporine, 2μg of cyclosporine H and 2μg of isocyclosporine H per 1ml.

[0112] Take cyclosporine eye gel, blank excipient, cyclosporine raw material respectively, adopt high temperature (121℃ for 4h), strong acid (1mol / L HCl, room temperature for 6h), strong base (1mol / L NaOH, room temperature for 5h), oxidation (30% hydrogen peroxide solution, room temperature for 5h), light (light intensity 4500±500Lux, ultraviolet lamp energy near ultraviolet lamp energy 0.65W·hr / m 2 , place 13d) severe conditions are destroyed.

[0113] The above solutions are respectively precisely measured, injected into a high performance liquid chromatograph, and detected according to the chromatographic conditions of Example 1, and the detection results are shown in Figure 5. As can be seen from Figure 5, the excipient has no obvious interference with the known impurity peaks of cyclosporine; the system suitability meets the requirements; the impurities generated by each degradation condition can be effectively separated; the cyclosporine raw material is unstable under acid and base conditions, and is relatively unstable under high temperature conditions, and different degrees of degradation are generated; the cyclosporine eye gel is only unstable under high temperature conditions, and the degradation impurities are mainly isocyclosporine A, isocyclosporine H and cyclosporine H. The related substance analysis method of the application has good specificity, and can effectively monitor the impurity detection of the product.

[0114] Example 4 linear and range experiment

[0115] Stock solution of reference substance: accurately weigh cyclosporine reference substance, cyclosporine B reference substance, cyclosporine C reference substance, cyclosporine D reference substance, cyclosporine H reference substance, cyclosporine U reference substance, dihydrocyclosporine A reference substance, isocyclosporine A reference substance, isocyclosporine H (trifluoroacetate) reference substance, dissolve in tetrahydrofuran and quantitatively dilute to prepare a stock solution containing about 0.1 mg of the above reference substances per 1 ml.

[0116] Linear stock solution: take 2.0 ml of each reference stock solution, place it in the same 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0117] 200% linear solution: accurately measure 4.0 ml of linear stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0118] 150% linear solution: accurately measure 3.0 ml of linear stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0119] 100% linear solution: accurately measure 2.0 ml of linear stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0120] 50% linear solution: accurately measure 1.0 ml of linear stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0121] 10% linear solution (limit of quantification): precisely pipette 1.0 ml of 100% linear solution into a 10-ml volumetric flask, dilute to the mark with diluent, shake well, and you get it.

[0122] Determination method: precisely pipette 100 μl of each concentration linear solution, inject into high performance liquid chromatograph, detect according to the chromatographic conditions in Example 1, and record the chromatogram. With the concentration (μg / ml) of linear solution as the abscissa X axis, the peak area (A) of each impurity as the ordinate Y axis, calculate the linear regression equation.

[0123] Correction factor calculation:

[0124] Take the slope (S) of each impurity linear function graph to calculate the correction factor of each impurity relative to cyclosporin and report. The calculation formula is as follows:

[0125] In the formula:

[0126] f: correction factor;

[0127] S 杂 : linear slope of each impurity;

[0128] S 环孢素(主峰) : linear slope of cyclosporin.

[0129] The linear investigation results are shown in Table 5, and the relative retention time and calculated correction factor of each impurity are shown in Table 6.

[0130] Table 5 Linear investigation results

[0131] The linear investigation results show that cyclosporin and each impurity are linear in the range of 200% of the limit of quantification (cyclosporin label amount 0.1%) to the impurity limit concentration (cyclosporin label amount 2.0%), the numerical value of each linear regression coefficient r is not less than 0.995, the ratio of each Y axis intercept to 100% response value is not greater than 10.0%, and the linear relationship is good.

[0132] Table 6 Relative retention time and calculated correction factor of each impurity

[0133] Example 5 Accuracy experiment

[0134] Impurity reference solution: precisely pipette 0.2 ml of each reference stock solution in Example 4 into a 10-ml volumetric flask, dilute to the mark with diluent.

[0135] Accuracy stock solution: take 2.0 ml of each impurity reference stock solution, put it in the same 20-ml volumetric flask, dilute to the mark with diluent, shake well, and you get it.

[0136] Blank adjuvant solution (base): accurately weigh 4 g of blank adjuvant (without cyclosporin), put into a 10 ml volumetric flask, add diluent, vortex mix uniformly, dissolve by ultrasonic, cool, add 0.2 ml of 10% calcium chloride solution, dilute to the mark with diluent, shake uniformly, centrifuge (10000 rpm, 10 min), take the supernatant as blank adjuvant solution.

[0137] 80% accuracy solution: accurately weigh 4 g of blank adjuvant, and accurately take 1.6 ml of accuracy stock solution, put into a 10 ml volumetric flask, add diluent, vortex mix uniformly, dissolve by ultrasonic (intensity 100, 15 min), cool, add 0.2 ml of 10% calcium chloride solution, dilute to the mark with diluent, shake uniformly, centrifuge (10000 rpm, 10 min), take the supernatant as spiked sample solution. Prepare 3 parallel samples.

[0138] 100% accuracy solution: accurately weigh 4 g of blank adjuvant, and accurately take 2.0 ml of accuracy stock solution, put into a 10 ml volumetric flask, add diluent, vortex mix uniformly, dissolve by ultrasonic (intensity 100, 15 min), cool, add 0.2 ml of 10% calcium chloride solution, dilute to the mark with diluent, shake uniformly, centrifuge (10000 rpm, 10 min), take the supernatant as spiked sample solution. Prepare 3 parallel samples.

[0139] 120% accuracy solution: accurately weigh 4 g of blank adjuvant, and accurately take 2.4 ml of accuracy stock solution, put into a 10 ml volumetric flask, add diluent, vortex mix uniformly, dissolve by ultrasonic (intensity 100, 15 min), cool, add 0.2 ml of 10% calcium chloride solution, dilute to the mark with diluent, shake uniformly, centrifuge (10000 rpm, 10 min), take the supernatant as spiked sample solution. Prepare 3 parallel samples.

[0140] Determination method:

[0141] Accurately take 100 μl of each of the above solutions, inject into high performance liquid chromatograph, detect according to the chromatographic conditions of Example 1, record the chromatogram, deduct the base, calculate the recovery rate of each sample and the RSD of 9 recovery rate results. The accuracy test results are shown in Table 7.

[0142] Table 7 Accuracy test results

[0143] The recovery rates of cyclosporin and each impurity are between 91.9% and 107.8%, and the RSDs are between 1.2 and 6.5%, which meet the requirements.

[0144] Example 6 Precision experiment

[0145] Stock solution of reference substance: precisely weigh 2 mg of cyclosporin, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin H, cyclosporin U, dihydrocyclosporin A, isocyclosporin A reference substance, 2.5 mg of isocyclosporin H (trifluoroacetate salt) respectively, and put them into 20 ml volumetric flasks, dissolve with tetrahydrofuran and dilute quantitatively to prepare stock solution containing about 0.1 mg of the above impurity reference substances per 1 ml. Take 2.0 ml of each impurity reference stock solution, and put them into the same 20 ml volumetric flask, dilute with diluent to the mark, and shake well to obtain the solution.

[0146] Test solution of repeatability: take 4 g of the product (about equivalent to 2 mg of cyclosporin), and precisely weigh and determine, and precisely take 2.0 ml of the stock solution of reference substance, and put them into a 10 ml volumetric flask, add an appropriate amount of diluent, vortex mix well, and then ultrasonic (intensity 100, 15 min) to dissolve, cool, add 0.2 ml of 10% calcium chloride solution, dilute with diluent to the mark, shake well, centrifuge (10000 rpm, 10 min), and take the supernatant as the spiked test solution. Prepare 6 samples in parallel.

[0147] Reference solution: precisely weigh an appropriate amount of cyclosporin reference substance, dissolve with diluent and dilute quantitatively to prepare a solution containing about 2 μg of cyclosporin per 1 ml.

[0148] Determination method: precisely take 100 μl of the above solution, inject into the liquid chromatograph, and detect according to the chromatographic conditions of Example 1, and record the chromatogram. Calculate the content of each impurity and its RSD in 6 samples. The results of repeatability test are shown in Table 8.

[0149] Table 8 Results of repeatability test

[0150] The results of repeatability test show that the RSD values of the content of each impurity in 6 test solution samples are not more than 10.0% (n=6), and the repeatability of the method is good.

[0151] Different personnel use different instruments to carry out tests at different times, and operate according to the same method under repeatability. The results of intermediate precision experiment are shown in Table 9.

[0152] Table 9 Results of intermediate precision verification

[0153] The results of intermediate precision verification show that the RSD values of the content of each impurity in 6 test solution samples are not more than 10.0% (n=6), and the intermediate precision meets the requirements; compared with the determination results under "repeatability", the RSD values of the content of each impurity are not more than 10.0% (n=12), and the precision of the method is good.

[0154] Experimental Example 7 Detection limit and quantification limit experiment

[0155] Quantification limit solution: an appropriate amount of the control substance stock solution under Example 4 was precisely measured, stepwise diluted with the diluent, and injected for detection to observe the signal-to-noise ratio of each impurity peak. When the signal-to-noise ratio was greater than 10, it was taken as the quantification limit solution.

[0156] Detection limit solution: 5.0 ml of the quantification limit solution was precisely measured and placed in a 10 ml volumetric flask, which was diluted to the calibration mark with the diluent and shaken to obtain the detection limit solution.

[0157] Determination method: 100 μl of the above solution was precisely measured and injected into the high performance liquid chromatograph, which was detected according to the chromatographic conditions of Example 1, and the chromatogram was recorded. The quantification limit solution was determined for 6 times continuously, and the detection limit solution was determined for 1 time. The determination results are shown in Table 10.

[0158] Table 10 Results of detection limit and quantification limit experiment

[0159] Experimental Example 8 Robustness experiment

[0160] The influence degree of the determination results when the column temperature, flow rate, wavelength, and chromatographic column in the chromatographic conditions were slightly changed was investigated. The variation parameters of the robustness experiment are shown in Table 11, and the rest of the chromatographic conditions were the same as those in Example 1. The experimental results are shown in Table 12.

[0161] Table 11 Variation parameters of the robustness experiment

[0162] Table 12 Results of the robustness system suitability investigation of each chromatographic condition

[0163] The above results show that under the conditions of different batches of chromatographic columns, wavelength 218 nm ± 2 nm, flow rate 0.9 ml / min to 1.2 ml / min, column temperature 58°C to 60°C, the impurity content detection results were within the range of normal conditions ± 0.1%, no new impurities were detected, and there was no significant influence on the detection results, indicating that the robustness of the method of the present application was good.

[0164] Experimental Example 9 Method comparison

[0165] The method before optimization (test (3) in Example 2) and the method after optimization (test (4) in Example 2) were used respectively for the related substance detection of the key batch of cyclosporine ophthalmic gel finished product, and the detection results are shown in Table 13.

[0166] Table 13 Comparison of related substance detection results before and after method optimization

[0167] The above results show that the related substance determination method of the cyclosporine eye gel provided in the application has stronger impurity detection capability under the condition of excluding the interference of excipients, and can effectively separate and detect cyclosporine U and dihydrocyclosporine A in the cyclosporine eye gel. Continuous sampling of multiple batches, stable chromatographic peak retention time, avoids overlapping of impurity positioning and inaccurate content determination caused by chromatographic peak drift. The application provides a solid and effective foundation for impurity research and control strategy of cyclosporine eye gel, thereby ensuring drug quality and safety.

[0168] The above is only a preferred specific embodiment of the present application; however, the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present application, according to the technical solution and improvement concept of the present application, makes equivalent replacement or change, which should be covered in the protection scope of the present application.

Claims

1. A method for detecting related substances in a cyclosporin product, characterized in that, The detection is performed by using high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography include: The chromatographic conditions of the high performance liquid chromatography further include at least one of the following: (1) the chromatographic column is Agilent AdvanceBio Peptide Map, 150mm*4.6mm, 2.7μm; 2. The detection method according to claim 1, characterized in that, (2) the concentration of phosphoric acid in the phosphoric acid aqueous solution is 0.05%-0.2%; (3) the flow rate is 0.9-1.2mL / min; (4) the column temperature is 58-60℃; (5) the injection volume is 50-100μL; (6) the detection wavelength is 216-220nm. The detection method further includes the step of preparing a test sample solution by using a test sample: taking the test sample, adding a diluent, dissolving, adding a calcium chloride solution, solid-liquid separation, and taking the liquid; Optionally, the diluent is a mixed solution of tetrahydrofuran and water, and the volume ratio of tetrahydrofuran to water is (3-4):(7-6); 3. The method of claim 1, wherein Optionally, the content of calcium chloride in the calcium chloride solution is 5%-20%; Optionally, the amount of the diluent is 8-10mL, and the amount of the calcium chloride solution is 0.1-0.5mL, relative to 2mg of cyclosporine; Optionally, the solid-liquid separation is centrifugation, and the centrifugal speed is 8000-12000rpm, and the centrifugal time is 5-20min. The detection method further includes the step of separately preparing a control sample solution by using a control sample: taking the control sample, adding a diluent, and dissolving. The control sample includes cyclosporine control sample, cyclosporine B control sample, cyclosporine C control sample, cyclosporine D control sample, cyclosporine H control sample, cyclosporine U control sample, dihydrocyclosporine A control sample, isocyclosporine A control sample, and isocyclosporine H control sample.

4. The method of claim 1, wherein, In the respective corresponding control sample solution, the concentration of the cyclosporine control sample is 2μg / mL, the concentration of the cyclosporine B control sample is 2μg / mL, the concentration of the cyclosporine C control sample is 2μg / mL, the concentration of the cyclosporine D control sample is 2μg / mL, the concentration of the cyclosporine H control sample is 2μg / mL, the concentration of the cyclosporine U control sample is 2μg / mL, the concentration of the dihydrocyclosporine A control sample is 2μg / mL, the concentration of the isocyclosporine A control sample is 2μg / mL, and the concentration of the isocyclosporine H control sample is 2μg / mL. ​ 5. The detection method according to claim 4, characterized in that, ​ 6. The detection method according to claim 4, characterized in that, The content of each related substance is calculated by the principal component external standard method with correction factor; The correction factor corresponding to isoridin H is 1.44, the correction factor corresponding to isoridin A is 1.19, the correction factor corresponding to cyclosporin C is 1.00, the correction factor corresponding to cyclosporin B is 1.32, the correction factor corresponding to cyclosporin U is 1.14, the correction factor corresponding to dihydro-cyclosporin A is 1.00, the correction factor corresponding to cyclosporin D is 1.13, the correction factor corresponding to cyclosporin H is 1.00, and the correction factor corresponding to unknown impurity is 1.

00.

7. The detection method according to any one of claims 1 to 6, characterized in that, The cyclosporin product includes cyclosporin raw material and cyclosporin preparation; Optionally, the cyclosporin preparation includes a preparation with polyoxyethylene castor oil as adjuvant.

8. The detection method according to claim 7, characterized in that, The cyclosporin preparation includes cyclosporin gel, and the cyclosporin gel uses polyoxyethylene castor oil as solubilizer.

9. The detection method according to claim 8, characterized in that, The cyclosporin gel includes the following components: Cyclosporin 0.05% to 0.1% (w / w), polyoxyethylene castor oil 0.95% to 1.05% (w / w), and gel base 0.04% to 0.10% (w / w).

10. Use of the detection method in any one of claims 1 to 9 in the quality control of cyclosporin product.

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