Detection method of vocyclosporine and derivatives thereof

Abstract

The invention relates to the technical field of high performance liquid chromatography, in particular to a high performance liquid chromatography detection method for vocyclosporine and derivatives thereof. According to the method, a chromatographic column takes porous graphitized carbon as a filling agent, a mobile phase A is one or more of acetonitrile and methanol, and a mobile phase B is one or more of isopropanol, methyl tert-butyl ether, tetrahydrofuran and 1, 4-dioxane; the separation degree between the Z / E-vocyclosporine isomers in the high performance liquid chromatography method is not less than 1.5. Compared with the existing nuclear magnetic method, the detection method provided by the invention is simple and easy to implement, is beneficial to reducing the production and detection cost, and is suitable for the related requirements of industrial and large-scale production; meanwhile, the method is used for controlling the quality of raw material medicines, intermediates or preparations and guaranteeing the medicine quality and safety, can be used for registration and declaration, and has great significance in research and development of imitated medicines and innovative medicines.

Description

Technical Field

[0001] The invention relates to the technical field of high performance liquid chromatography, and in particular to a high performance liquid chromatography detection method for cyclosporine and its derivatives. Background Art

[0002] Cyclosporin derivatives (a class of cyclic polypeptides produced by the fungus Tolypocladium inflatum Gams) are increasingly used in immunosuppressive therapy because they primarily act on T-cell-mediated responses. Cyclosporin derivatives have been observed to reversibly suppress immunocompetent lymphocytes, especially T-lymphocytes, and inhibit the production and release of lymphokines. Both cyclosporine and acetylcyclosporine are cyclosporin analogs, both of which have a Z / E configuration at the double bond.

[0003]

[0004] The existing detection of the isomer ratio of Z / E-cyclosporine mainly relies on nuclear magnetic resonance (NMR). The detection instruments involved are relatively expensive and generally not available in research institutions or enterprises. The samples need to be sent to professional testing institutions for testing, which is very inconvenient. At the same time, the test results of the samples have a certain lag, which is not conducive to the quality control of the production process. In addition, the ratio of Z-configuration isomers in Z / E-cyclosporine is usually low (<5%), and the nuclear magnetic resonance method cannot pass the methodological verification, so it is also very inconvenient at the drug registration level.

[0005] In patents CN1571794A (published on 2005-01-26) and CN1571795A (published on 2005-01-26), the percentage content of one isomer or another isomer in a mixture is confirmed by nuclear magnetic resonance spectroscopy (NMR). Patent CN1798759A (published on 2006-07-05) also reported that the purity of E-cyclosporine is 99% by HPLC analysis, but the HPLC detection method was not specifically disclosed.

[0006] Therefore, the development of chromatographic methods is of great significance for both quality control of the product production process and product registration declaration. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 : HPLC spectrogram of Example 1

[0008] Figure 2 : HPLC spectrogram of embodiment 2

[0009] Figure 3 : HPLC spectrogram of embodiment 3

[0010] Figure 4 : HPLC spectrogram of embodiment 4

[0011] Figure 5 : HPLC spectrogram of Comparative Example 1

[0012] Figure 6 : HPLC spectrogram of Comparative Example 2 Summary of the invention

[0013] In order to solve the above problems, the present invention provides a method for detecting cyclosporine and its derivatives, and the specific scheme is as follows:

[0014] A method for detecting cyclosporine and its derivatives, the method comprising detecting by high performance liquid chromatography, wherein the liquid chromatography conditions are:

[0015] Chromatographic column: porous graphene as filler;

[0016] Mobile phase: Mobile phase A is one or more of acetonitrile or methanol, and mobile phase B is one or more of isopropanol, methyl tert-butyl ether, tetrahydrofuran, or 1,4-dioxane;

[0017] In the preferred technical solution of the present invention, the flow rate of the mobile phase is 0.5-1.5 mL / min, preferably 1.0 mL / min;

[0018] In the preferred technical solution of the present invention, the column temperature is 70-100°C, preferably 80-90°C;

[0019] In the preferred technical solution of the present invention, the injection volume is 1-20 μL, preferably 1-10 μL;

[0020] In the preferred technical solution of the present invention, the detection wavelength is 210nm-237nm, preferably any one of 210nm and 237nm;

[0021] In the preferred technical scheme of the present invention, mobile phase A is acetonitrile, and mobile phase B is one of methyl tert-butyl ether or tetrahydrofuran, wherein the volume ratio of mobile phase A:mobile phase B is 15:85 to 85:15, preferably 25:75 to 75:25, and more preferably 35:65 to 65:35.

[0022] In the preferred technical scheme of the present invention, mobile phase A is acetonitrile, and mobile phase B is methyl tert-butyl ether and tetrahydrofuran, wherein the volume ratio of mobile phase A: mobile phase B is 20:80-80:20, preferably 30:70-70:30, and more preferably 40:60; wherein the volume ratio of methyl tert-butyl ether and tetrahydrofuran in mobile phase B is 8:1-15:1, preferably 9:1-12:1, and more preferably 11:1.

[0023] In the preferred technical scheme of the present invention, the chromatographic column is a Thermo HYPERCARB chromatographic column, the specifications of the chromatographic column are any one of 100*2.1mm, 100*4.6mm, 150*3mm, 150*4.6mm, 250*3mm, and 250*4.6mm, and the particle size of the chromatographic column filler is any one of 3μm and 5μm.

[0024] In a preferred technical solution of the present invention, the liquid chromatography conditions also include an elution mode, and the elution mode is isocratic elution.

[0025] To further optimize the detection method, when the mobile phase A is acetonitrile and the mobile phase B is methyl tert-butyl ether, the following elution procedure is used for elution:

[0026]

[0027] To further optimize the detection method, when the mobile phase A is acetonitrile and the mobile phase B is tetrahydrofuran, the following elution procedure is used for elution:

[0028]

[0029] To further optimize the detection method, when the mobile phase A is acetonitrile and the mobile phase B is methyl tert-butyl ether and tetrahydrofuran, the following elution procedure is used for elution:

[0030]

[0031] In a preferred technical solution of the present invention, the separation degree of Z / E-cyclosporine isomers is ≥0.9, preferably ≥1.0, and more preferably ≥1.5.

[0032] Another object of the present invention is to provide an application of the high performance liquid chromatography method of the present invention in separating and purifying cyclosporine and its derivatives.

[0033] Another object of the present invention is to provide an application of the method for separating and detecting cyclosporine and its derivatives and / or its related substances by high performance liquid chromatography in the quality control of cyclosporine and its derivatives raw materials and / or their preparations.

[0034] In a preferred technical solution of the present invention, the preparation of cyclosporine and its derivatives is selected from solid preparations or liquid preparations of cyclosporine and its derivatives.

[0035] In the preferred technical solution of the present invention, the solid preparation of cyclosporine and its derivatives is selected from any one of tablets, capsules, powders, granules, dry suspensions and pellets.

[0036] In the preferred technical solution of the present invention, the liquid preparation of cyclosporine and its derivatives is selected from any one of oral liquid, syrup, injection, spray and suspension.

[0037] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0038] Beneficial Effects

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) Compared with the existing nuclear magnetic resonance method for detecting the ratio of Z / E-cyclosporine isomers, the detection method provided by the present invention is simple and easy to use, which is conducive to reducing the production and detection costs and is suitable for the relevant requirements of industrial and large-scale production;

[0041] (2) The separation degree between Z / E-cyclosporine isomers in the high performance liquid chromatography method of the present invention is not less than 1.5, and can be used for separation, content determination, preparation and purification of cyclosporine or its derivatives, related substances or monitoring of its related substances;

[0042] (3) Compared with the existing nuclear magnetic resonance method, the detection method provided by the present invention can be verified by methodology, used to control the quality of raw materials, intermediates or preparations, ensure the quality and safety of drugs, and can be used for registration and declaration, which is of great significance to the research and development of generic drugs and innovative drugs. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0044] Resolution (R): The ratio of the retention time of two adjacent peaks to the average peak width, also known as resolution, which indicates the degree of separation of two adjacent peaks. The larger R is, the better the separation of two adjacent components.

[0045] The content of Z-acetylcyclosporine in the present invention is calculated using the following formula:

[0046]

[0047] Among them A Z-伏环孢素 is the peak area of ​​Z-cyclosporine, A E-伏环孢素 is the peak area of ​​E-cyclosporine, f Z-伏环孢素is the relative response factor of Z-cyclosporine, 1.0, and P is the conversion rate of acetylcyclosporine to cyclosporine (the experimental value is 75%).

[0048] The content of Z-cyclosporine in the present invention is calculated using the following formula:

[0049]

[0050] Among them A Z-伏环孢素 is the peak area of ​​Z-cyclosporine, A E-伏环孢素 is the peak area of ​​E-cyclosporine, f Z-伏环孢素 The relative response factor of Z-cyclosporine is 1.0

[0051] Example 1

[0052] Sample preparation: Accurately weigh 200 mg of acetylcyclosporine sample and place it in a 20 mL volumetric flask. Accurately add 4 mL of methanol and 1 mL of water. Mix and dissolve, then add 0.19 g of potassium carbonate. Shake in a 50°C water bath for 3 hours, then cool to room temperature, transfer all to a 200 mL volumetric flask, and dilute to the mark with methanol.

[0053] Testing process: Analyze the sample in the above volumetric flask using HPLC method. Directly inject the solution for analysis. Testing conditions are as follows:

[0054] Instrument: High Performance Liquid Chromatograph Agilent 1260

[0055] Chromatographic column: Thermo Hypercarb (150 mm × 4.6 mm, 3 μm), porous graphite carbon filler;

[0056] Detector: UV detector;

[0057] Detection wavelength: 237nm;

[0058] Mobile phase: acetonitrile / methyl tert-butyl ether / tetrahydrofuran = 40:55:5;

[0059] Flow rate: 1mL / min,

[0060] Injection volume: 10 μL;

[0061] Column temperature: 85°C;

[0062] Elution method: isocratic elution (see Table 1).

[0063] Table 1: Elution conditions of Example 1

[0064]

[0065] The results are as follows Figure 1As shown, 13min is Z-configuration cyclosporine, and the content of Z-configuration acetylcyclosporine is 2% after conversion; 14min is E-configuration cyclosporine, and the content of E-configuration acetylcyclosporine is 98% after conversion. The main component peak is obvious, the column pressure does not change significantly, and the separation degree is 1.51, indicating a good separation effect.

[0066] Example 2

[0067] Sample preparation: Same as Example 1.

[0068] Testing process: Analyze the sample in the above volumetric flask using HPLC method. Directly inject the solution for analysis. Testing conditions are as follows:

[0069] Instrument: High Performance Liquid Chromatograph Agilent 1260

[0070] Chromatographic column: Thermo Hypercarb (150 mm × 4.6 mm, 3 μm);

[0071] Detector: UV detector;

[0072] Detection wavelength: 210nm;

[0073] Mobile phase: acetonitrile / methyl tert-butyl ether = 35:65;

[0074] Flow rate: 1mL / min,

[0075] Injection volume: 5 μL;

[0076] Column temperature: 85°C;

[0077] Elution method: isocratic elution (see Table 2).

[0078] Table 2: Elution conditions of Example 2

[0079]

[0080] The results are as follows Figure 2 As shown, 15min is Z-configuration cyclosporine, and the content of Z-configuration acetylcyclosporine is 42% after conversion; 17min is E-configuration cyclosporine, and the content of E-configuration acetylcyclosporine is 58% after conversion. The main component peak is obvious, the column pressure does not change significantly, and the separation degree is 2.1, indicating a good separation effect.

[0081] Example 3

[0082] Sample preparation: Same as Example 1.

[0083] Testing process: Analyze the sample in the above volumetric flask using HPLC method. Directly inject the solution for analysis. Testing conditions are as follows:

[0084] Instrument: High Performance Liquid Chromatograph Agilent 1260

[0085] Chromatographic column: Thermo Hypercarb (150 mm × 4.6 mm, 3 μm);

[0086] Detector: UV detector;

[0087] Detection wavelength: 210nm;

[0088] Mobile phase: tetrahydrofuran / acetonitrile = 35:65;

[0089] Flow rate: 1mL / min,

[0090] Injection volume: 5 μL;

[0091] Column temperature: 85°C;

[0092] Elution method: isocratic elution (see Table 3).

[0093] Table 3: Elution conditions of Example 3

[0094]

[0095] The results are as follows Figure 3 As shown, 13min is Z-configuration cyclosporine, and the content of Z-configuration acetylcyclosporine is 42% after conversion; 15min is E-configuration cyclosporine, and the content of E-configuration acetylcyclosporine is 58% after conversion. The main component peak is obvious, the column pressure does not change significantly, and the separation degree is 1.7, indicating a good separation effect.

[0096] Example 4

[0097] Sample preparation: Take an appropriate amount of cyclosporine test sample and reference sample, weigh accurately, add methanol to dissolve and dilute to make a solution containing about 1 mg of cyclosporine per 1 mL.

[0098] Testing process: Analyze the sample in the above volumetric flask using HPLC method. Directly inject the solution for analysis. Testing conditions are as follows:

[0099] Instrument: High Performance Liquid Chromatograph Agilent 1260

[0100] Chromatographic column: Thermo Hypercarb (150 mm × 4.6 mm, 3 μm);

[0101] Detector: UV detector;

[0102] Detection wavelength: 237nm;

[0103] Mobile phase: acetonitrile / methyl tert-butyl ether / tetrahydrofuran = 40:55:5;

[0104] Flow rate: 1mL / min,

[0105] Injection volume: 10 μL;

[0106] Column temperature: 85°C;

[0107] Elution method: isocratic elution (see Table 4).

[0108] Table 4: Elution conditions of Example 4

[0109]

[0110] The test results of this embodiment are as follows Figure 4 As shown, 12min is Z-configuration cyclosporine, with a content of 55%; 14min is E-configuration cyclosporine, with a content of 45%. The main component peak is obvious, the column pressure has no obvious change, and the separation degree is 2.14, indicating a good separation effect.

[0111] Comparative Example 1

[0112] Sample preparation: accurately weigh 200 mg of acetylcyclosporine sample and place it in a 20 mL volumetric flask, accurately add 4 mL of acetonitrile and 1 mL of water, mix and dissolve, then add 0.19 g of potassium carbonate, shake in a 50°C water bath for 3 hours, then cool to room temperature, transfer all to a 200 mL volumetric flask, and dilute to the scale with acetonitrile.

[0113] Testing process: Analyze the sample in the above volumetric flask using HPLC method. Directly inject the solution for analysis. Testing conditions are as follows:

[0114] Instrument: High Performance Liquid Chromatograph Agilent 1260

[0115] Chromatographic column: Agilent Zorbax SB-CN (3.0×100 mm, 1.8 μm), cyano column;

[0116] Detector: UV detector;

[0117] Detection wavelength: 210nm;

[0118] Mobile phase: 0.1% phosphoric acid solution / acetonitrile;

[0119] Flow rate: 0.5mL / min,

[0120] Injection volume: 2 μL;

[0121] Column temperature: 70°C;

[0122] Elution mode: gradient elution (see Table 5).

[0123] Table 5: Elution conditions of Comparative Example 1

[0124]

[0125] The results are as follows Figure 5 As shown, there is only one major peak and this method has no resolution for this substrate.

[0126] Comparative Example 2

[0127] Sample preparation: accurately weigh 200 mg of acetylcyclosporine sample and place it in a 20 mL volumetric flask, accurately add 4 mL of acetonitrile and 1 mL of water, mix and dissolve, then add 0.19 g of potassium carbonate, shake in a 50°C water bath for 3 hours, then cool to room temperature, transfer all to a 200 mL volumetric flask, and dilute to the scale with acetonitrile.

[0128] Testing process: Analyze the sample in the above volumetric flask using HPLC method. Directly inject the solution for analysis. Testing conditions are as follows:

[0129] Instrument: High Performance Liquid Chromatograph Agilent 1260

[0130] Chromatographic column: Waters XSelect HSS PFP (150 mm × 3.0 mm, 2.5 μm), pentafluorophenyl column;

[0131] Detector: UV detector;

[0132] Detection wavelength: 210nm;

[0133] Mobile phase: 0.1% phosphoric acid solution / acetonitrile;

[0134] Flow rate: 0.5mL / min,

[0135] Injection volume: 5 μL;

[0136] Column temperature: 50°C;

[0137] Elution mode: gradient elution (see Table 6).

[0138] Table 6: Elution conditions of Comparative Example 2

[0139]

[0140] The results are as follows Figure 6 As shown, there is only one major peak and this method has no resolution for this substrate.

[0141] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and controls can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for detecting cyclosporine and its derivatives, characterized in that: The method comprises detecting by high performance liquid chromatography, wherein the liquid chromatography conditions are: Chromatographic column: porous graphene as filler; Mobile phase: Mobile phase A is one or more of acetonitrile or methanol, and mobile phase B is one or more of isopropanol, methyl tert-butyl ether, tetrahydrofuran, or 1,4-dioxane.

2. The detection method according to claim 1, characterized in that The liquid chromatography conditions also include: The mobile phase flow rate is 0.5-1.5 mL / min, preferably 1.0 mL / min; The column temperature is 70-100°C, preferably 80-90°C; The injection volume is 1-20 μL, preferably 1-10 μL; and / or The detection wavelength is 210 nm-237 nm, preferably 210 nm or 237 nm.

3. The detection method according to claim 1, characterized in that The liquid chromatography conditions also include: the elution mode is isocratic elution.

4. The detection method according to any one of claims 1 to 3, characterized in that: The mobile phase A is acetonitrile, and the mobile phase B is one of methyl tert-butyl ether and tetrahydrofuran, wherein the volume ratio of mobile phase A to mobile phase B is 15:85 to 85:15, preferably 25:75 to 75:25, and more preferably 35:65 to 65:

35.

5. The detection method according to any one of claims 1 to 3, characterized in that: The mobile phase A is acetonitrile, and the mobile phase B is methyl tert-butyl ether and tetrahydrofuran, wherein the volume ratio of mobile phase A:mobile phase B is 20:80-80:20, preferably 30:70-70:30, and more preferably 40:60; wherein the volume ratio of methyl tert-butyl ether to tetrahydrofuran in mobile phase B is 8:1-15:1, preferably 9:1-12:1, and more preferably 11:

1.

6. The detection method according to any one of claims 1 to 3, characterized in that: The chromatographic column is a ThermoHYPERCARB chromatographic column, and the specifications of the chromatographic column are any one of 100*2.1mm, 100*4.6mm, 150*3mm, 150*4.6mm, 250*3mm, and 250*4.6mm, and the particle size of the chromatographic column filler is any one of 3μm and 5μm.

7. The detection method according to any one of claims 1 to 3, characterized in that: The separation degree of Z / E-cyclosporine isomers under the chromatographic conditions is ≥0.9, preferably ≥1.0, and more preferably ≥1.

5.

8. Use of the detection method according to any one of claims 1 to 7 in separating and purifying cyclosporine and its derivatives.

9. Use of the detection method according to any one of claims 1 to 8 in the quality control of cyclosporine and its derivative raw materials or preparations thereof.

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