Method for detecting related substances of aminophylline injection

Abstract

A method for detecting related substances of an aminophylline injection belongs to the field of pharmaceutical analysis, and adopts a high performance liquid chromatography for detection, and the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filler in a chromatographic column; a mobile phase A is an ammonium acetate buffer solution; a mobile phase B is methanol; the volume ratio of the mobile phase A to the mobile phase B is (70-90): (10-30). According to the method, the aminophylline and the related substances are qualitatively or quantitatively detected by adopting high performance liquid chromatography, and the aminophylline injection and the related substances are good in linear relation, good in accuracy and precision, high in specificity and high in stability. The method is good in reproducibility, can meet the detection requirements of the related substances of the aminophylline injection, and can be used for quality control of the aminophylline injection.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting related substances of aminophylline injection. Background Art

[0002] Aminophylline is a commonly used drug for the clinical treatment of asthma, and its main dosage forms are tablets and injections. Aminophylline has a narrow therapeutic index, with an effective blood concentration range of 10μg / mL-20μg / mL. If its effective blood concentration is higher than 20μg / mL, toxic reactions are likely to occur, and in severe cases, respiratory and cardiac arrest may lead to death. When aminophylline is used clinically as an injection, it generally needs to be slowly pushed in, and the blood concentration must be monitored at all times.

[0003] At present, there is no report on the high performance liquid chromatography detection method for aminophylline injection related substances. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting related substances of aminophylline injection.

[0005] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0006] A method for detecting related substances of aminophylline injection of the present invention adopts high performance liquid chromatography for detection, and the chromatographic conditions of the high performance liquid chromatography are:

[0007] The chromatographic column uses octadecylsilane bonded silica as filler;

[0008] Mobile phase A: ammonium acetate buffer;

[0009] Mobile phase B: methanol;

[0010] The volume ratio of the mobile phase A to the mobile phase B is (70-90):(10-30).

[0011] As a preferred embodiment, the chromatographic column is Agilent ZORBAX Eclipse Plus C18, and its specifications are 4.6×250 mm, 5 μm.

[0012] As a preferred embodiment, the preparation process of the mobile phase A is as follows:

[0013] Place 770.8 mg of ammonium acetate in a 1000 ml beaker and dissolve in 800 ml of water. Adjust the pH to 5.5 with glacial acetic acid and dilute with water to 1000 ml.

[0014] As a preferred embodiment, in the high performance liquid chromatography method, the detection wavelength is 270 nm, the flow rate is 1.0 ml / min, the column temperature is 30° C., and the injection volume is 20 μl.

[0015] As a preferred embodiment, in the high performance liquid chromatography, gradient elution is performed according to the following table:

[0016] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 86-90 10-14 2 86-90 10-14 20 70 30 25-30 70 30 25.1-30.1 86-90 10-14 40-45 86-90 10-14

[0017] As a more preferred embodiment, in the high performance liquid chromatography, gradient elution is performed according to the following table:

[0018] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 2 90 10 20 70 30 30 70 30 30.1 90 10 45 90 10

[0019] As a preferred embodiment, the related substances are:

[0020] Impurity A: 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione;

[0021] Impurity B: 3-methyl-3,7-dihydro-1H-purine-2,6-dione;

[0022] Impurity C: N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)formamide;

[0023] Impurity D: N-methyl-5-(methylamino)-1H-imidazole-4-carboxamide hydrochloride monohydrate;

[0024] Impurity E: 1,3-dimethyl-7,9-dihydro-1H-purine-2,6,8(3H)-trione;

[0025] Impurity F: 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione;

[0026] Impurity G: 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione;

[0027] Theophylline.

[0028] The beneficial effects of the present invention are:

[0029] The invention discloses a method for detecting related substances of aminophylline injection, which uses high performance liquid chromatography to perform qualitative or quantitative detection on aminophylline and related substances, thus filling the blank of qualitative or quantitative detection of related substances of aminophylline injection. The invention discloses a method for detecting related substances of aminophylline injection, which has good linear relationship between aminophylline injection and related substances, good accuracy and precision, strong specificity and high stability.

[0030] The method for detecting related substances of aminophylline injection of the present invention has good reproducibility, can meet the detection requirements of related substances of aminophylline injection, and can be used for quality control of aminophylline injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the chromatogram of the spiked test solution in Example 1.

[0032] Figure 2 This is the chromatogram of the spiked test solution in Example 2 (the chromatographic column is CPACELLPAK-C18 (4.6 mm×250 mm, 5 μm)).

[0033] Figure 3 This is the chromatogram of the spiked test solution in Example 2 (the chromatographic column is Agilent ZORBAX EclipsePlus C18 (4.6×250 mm, 5 μm)).

[0034] Figure 4 This is the chromatogram of the spiked test solution in Example 3.

[0035] Figure 5 This is the chromatogram of the spiked test solution in Example 4. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1 Optimization of detection conditions for related substances of aminophylline injection

[0038] (1) The chromatographic conditions are as follows:

[0039] Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm);

[0040] Detection wavelength: 270nm;

[0041] Flow rate: 1.0ml / min;

[0042] Column temperature: 30°C;

[0043] Injection volume: 20 μl;

[0044] Mobile phase A: Place 770.8 mg of ammonium acetate in a 1000 ml beaker and dissolve in 800 ml of water. Adjust the pH to 5.5 with glacial acetic acid and dilute to 1000 ml with water.

[0045] Mobile phase B: methanol;

[0046] Then, gradient elution was performed according to Table 1 to optimize the gradient conditions.

[0047] Table 1 Gradient elution parameters

[0048] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 86 14 2 86 14 20 70 30 25 70 30 25.1 86 14 40 86 14

[0049] (2) Preparation of solution;

[0050] (2-1) Preparation of each impurity localization solution: Take appropriate amount of each impurity A reference substance, impurity C reference substance, impurity D reference substance, impurity F reference substance and impurity G reference substance, add appropriate amount of methanol to dissolve them, then add a mixed solution of methanol and water (the volume ratio of methanol to water is 1:6) to dilute them to a suitable concentration, which is used as the localization solution of each impurity A, impurity C, impurity D, impurity F and impurity G; take appropriate amount of impurity B reference substance and impurity E reference substance, dissolve them in 0.5 ml of 0.1 mol / L sodium hydroxide, then add a mixed solution of methanol and water (the volume ratio of methanol to water is 1:6) to dilute them to a suitable concentration, which is used as the localization solution of impurities B and impurity E. The concentration of each impurity localization solution is 10 μg / ml.

[0051] (2-2) Preparation of spiked test solution: Take 25 mg of aminophylline API, accurately weigh it, and place it in a 10 ml volumetric flask. Add each impurity localization solution, with the amount of each impurity localization solution added being 1 ml. Dissolve it in a mixed solution of methanol and water (the volume ratio of methanol to water is 1:6) and quantitatively dilute it to prepare the spiked test solution.

[0052] Among them, the formula and preparation method of the aminophylline raw material used are as follows:

[0053] Weigh 250 mg of aminophylline and 3 mg of ethylenediamine respectively, add water for injection to 20 ml, stir until completely dissolved, then filter, fill into vials or ampoules, and sterilize by wet heat at 121°C for 15 minutes.

[0054] (3) Determination;

[0055] Accurately measure 20 μl of the spiked test solution, inject it into the liquid chromatograph for testing (determined according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512)), and record the chromatogram.

[0056] The chromatogram of the spiked test solution is as follows Figure 1As shown, the tailing factor of the main peak (theophylline peak) is 3.115. The tailing of the main peak interferes with impurity F, resulting in the main peak and impurity F not being separated. The chromatographic conditions are further optimized by replacing the chromatographic column.

[0057] Example 2 Optimization of detection conditions for related substances of aminophylline injection

[0058] (1) The chromatographic conditions are as follows:

[0059] Replace the chromatographic columns: CPACELL PAK-C18 (4.6 mm × 250 mm, 5 μm) and Agilent ZORBAX Eclipse Plus C18 (4.6 × 250 mm, 5 μm);

[0060] Other chromatographic conditions were the same as in Example 1.

[0061] (2) Preparation of solution;

[0062] Same as Example 1.

[0063] (3) Determination;

[0064] Accurately measure 20 μl of the spiked test solution, inject it into the liquid chromatograph for testing (determined according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512)), and record the chromatogram.

[0065] The chromatogram of the spiked test solution is as follows Figure 2 (The chromatographic column is CPACELL PAK-C18 (4.6 mm × 250 mm, 5 μm)), Figure 3 (The chromatographic column is Agilent ZORBAX Eclipse Plus C18 (4.6×250mm, 5μm)) and Table 2. Using the CPACELLPAK-C18 column, the known impurity D and impurity E are not completely separated; the tailing factor of theophylline peak when using the Agilent ZORBAXEclipse Plus column is 1.897, and the separation between each known impurity is greater than 2.0, but it can be seen from the chromatogram that the tailing of theophylline peak interferes with impurity F, resulting in the main peak and impurity F not being separated, but the chromatographic column conditions are better than the CPACELLPAK-C18 column, so the Agilent ZORBAX Eclipse Plus column conditions are better than the CPACELLPAK-C18 column; by reducing the methanol ratio in the initial gradient, extending the retention time, and improving the separation effect between each impurity, the chromatographic conditions are further optimized.

[0066] Table 2 Analysis results of spiked test solution

[0067]

[0068] Example 3 Optimization of detection conditions for related substances of aminophylline injection

[0069] (1) The chromatographic conditions are as follows:

[0070] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 μm);

[0071] Detection wavelength: 270nm;

[0072] Flow rate: 1.0ml / min;

[0073] Column temperature: 30°C;

[0074] Injection volume: 20 μl;

[0075] Mobile phase A: Place 770.8 mg of ammonium acetate in a 1000 ml beaker and dissolve in 800 ml of water. Adjust the pH to 5.5 with glacial acetic acid and dilute to 1000 ml with water.

[0076] Mobile phase B: methanol;

[0077] Then, gradient elution was performed according to Table 3 to optimize the gradient conditions.

[0078] Table 3

[0079] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 2 90 10 20 70 30 25 70 30 25.1 90 10 40 90 10

[0080] (2) Preparation of solution;

[0081] (2-1) Prepare each impurity location solution: take appropriate amounts of each impurity A reference substance (1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione), impurity C reference substance (N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)carboxamide), impurity D reference substance (N-methyl-5-(methylamino)-1H-imidazole-4-carboxamide monohydrate hydrochloride), impurity F reference substance (7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione) and impurity G reference substance (3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione), respectively. After adding an appropriate amount of methanol to dissolve, a mixed solution of methanol and water (the volume ratio of methanol to water is 1:6) is added to dilute to a suitable concentration, which is used as the location solution of each impurity A, impurity C, impurity D, impurity F and impurity G; appropriate amounts of impurity B reference substance (3-methyl-3,7-dihydro-1H-purine-2,6-dione) and impurity E reference substance (1,3-dimethyl-7,9-dihydro-1H-purine-2,6,8(3H)-trione) are taken, respectively, dissolved with 0.5 ml of 0.1 mol / L sodium hydroxide, and then a mixed solution of methanol and water (the volume ratio of methanol to water is 1:6) is added to dilute to a suitable concentration, which is used as the location solution of impurities B and impurity E. Among them, the concentration of each impurity location solution is 10 μg / ml.

[0082] (2-2) Preparation of spiked test solution: Take 25 mg of aminophylline API, accurately weigh it, place it in a 10 ml volumetric flask, add each impurity localization solution, and add 2 ml of each impurity localization solution. Dissolve it in a mixed solution of methanol and water (the volume ratio of methanol to water is 1:6) and quantitatively dilute it to prepare the spiked test solution.

[0083] Among them, the formula and preparation method of the aminophylline raw material used are as follows:

[0084] Weigh 250 mg of aminophylline and 3 mg of ethylenediamine respectively, add water for injection to 20 ml, stir until completely dissolved, then filter, fill into vials or ampoules, and sterilize by wet heat at 121°C for 15 minutes.

[0085] (3) Determination;

[0086] Accurately measure 20 μl of the spiked test solution, inject it into the liquid chromatograph for testing (determined according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512)), and record the chromatogram.

[0087] The chromatogram of the spiked test solution is as follows Figure 4As shown in Table 4. Using the Agilent ZORBAX Eclipse Plus column, in the chromatogram of the spiked test solution, the tailing factor of theophylline peak was 1.733, and the separation between each known impurity was greater than 2.0; however, it can be seen from the chromatogram that the tailing of theophylline peak interfered with impurity F, resulting in the main peak and impurity F not being completely separated; by adjusting the gradient running time, the gradient was made gentle, the separation between each impurity was improved, and the chromatographic conditions were further optimized.

[0088] Table 4 Analysis results of spiked test solution

[0089]

[0090]

[0091] Example 4 Optimization of detection conditions for related substances of aminophylline injection

[0092] (1) The chromatographic conditions are as follows:

[0093] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 μm);

[0094] Detection wavelength: 270nm;

[0095] Flow rate: 1.0ml / min;

[0096] Column temperature: 30°C;

[0097] Injection volume: 20 μl;

[0098] Mobile phase A: Place 770.8 mg of ammonium acetate in a 1000 ml beaker and dissolve in 800 ml of water. Adjust the pH to 5.5 with glacial acetic acid and dilute to 1000 ml with water.

[0099] Mobile phase B: methanol;

[0100] Then, gradient elution was performed according to Table 5 to optimize the gradient conditions.

[0101] Table 5

[0102] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 2 90 10 20 70 30 30 70 30 30.1 90 10 45 90 10

[0103] (2) Preparation of solution;

[0104] Same as Example 3.

[0105] (3) Determination;

[0106] Accurately measure 20 μl of the spiked test solution, inject it into the liquid chromatograph for testing (determined according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512)), and record the chromatogram.

[0107] The chromatogram of the spiked test solution is as follows Figure 5 As shown in Table 6. Under the chromatographic conditions, in the chromatogram of the spiked test solution, the retention time of theophylline peak is 16.028min, the tailing factor of theophylline peak is 1.815, does not interfere with impurity F, and the separation degree between the main peak and impurity F is 5.608, which meets the requirements; the separation degree between each known impurity is greater than 2.0, and the symmetry of the main peak, retention time and separation degree between impurities are better than other chromatographic methods. The chromatographic conditions used in this embodiment are determined to be the final chromatographic conditions of the method for detecting related substances of aminophylline injection.

[0108] Table 6 Analysis results of spiked test solution

[0109] Impurity Name Retention time Peak tailing factor Separation Theoretical plates Impurity C 4.220 1.119 -- 1364 Impurity B 7.698 0.974 9.475 12374 Impurity D 8.736 0.956 3.667 14561 Impurity E 9.572 0.985 2.750 14484 Impurity G 11.681 0.955 7.262 31581 Theophylline 16.028 1.815 12.508 22014 Impurity F 17.988 0.976 5.608 72827 Impurity A 21.014 0.994 11.123 91739

[0110] Example 5 Detection Method of Related Substances in Aminophylline Injection

[0111] (1) The chromatographic conditions are as follows:

[0112] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 μm);

[0113] Detection wavelength: 270nm;

[0114] Flow rate: 1.0ml / min;

[0115] Column temperature: 30°C;

[0116] Injection volume: 20 μl;

[0117] Mobile phase A: Place 770.8 mg of ammonium acetate in a 1000 ml beaker and dissolve in 800 ml of water. Adjust the pH to 5.5 ± 0.05 with glacial acetic acid, dilute to 1000 ml with water and shake well.

[0118] Mobile phase B: methanol;

[0119] Then perform linear gradient elution according to Table 7.

[0120] Table 7

[0121] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 2 90 10 20 70 30 30 70 30 30.1 90 10 45 90 10

[0122] (2) Preparation of solution:

[0123] (2-1) Preparation of test solution: Take an appropriate amount of aminophylline raw material (equivalent to 25 mg of aminophylline) and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0124] Among them, the formula and preparation method of the aminophylline raw material used are as follows:

[0125] Weigh 250 mg of aminophylline and 3 mg of ethylenediamine respectively, add water for injection to 20 ml, stir until completely dissolved, then filter, fill into vials or ampoules, and sterilize by wet heat at 121°C for 15 minutes.

[0126] (2-2) Prepare control solution: Accurately measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Add solvent to the mark. Shake well and accurately transfer 1 ml to a 10 ml volumetric flask. Dilute to the mark with solvent (methanol-water (10:90)) and shake well.

[0127] (2-3) Preparation of system suitability solution: Take an appropriate amount of impurity F reference substance [7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione], add an appropriate amount of water and sonicate to dissolve it, dilute with solvent (methanol-water (10:90)) to prepare a stock solution of impurity F containing approximately 20 μg per ml. Take an appropriate amount of theophylline reference substance, add an appropriate amount of solvent and sonicate to dissolve it, add an appropriate amount of impurity F stock solution, and dilute with solvent (methanol-water (10:90)) to prepare a solution containing approximately 2.5 mg of theophylline and approximately 2 μg of impurity F per ml.

[0128] (2-4) System suitability requirements: In the system suitability solution chromatogram, the number of theoretical plates calculated based on theophylline peak should be no less than 5000, and the separation between theophylline peak and impurity F peak should be no less than 2.0.

[0129] Test Example 1 Methodological Verification of the Detection Method of Aminophylline Injection Related Substances of the Present Invention

[0130] According to the guidance principles for validation of analytical methods of drug quality standards, the specificity, linearity and range, quantitative limit and detection limit, impurity correction factor, recovery rate, precision, solution stability and durability of the detection method of the aminophylline injection related substances of the present invention were validated. The specific methodological validation results are summarized in Table 8.

[0131] Table 8 Methodological verification results of related substances

[0132]

[0133]

[0134] The present invention discloses a method for detecting related substances of aminophylline injection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The product of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the product described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

Claims

1. A method for detecting related substances of aminophylline injection, characterized in that: The detection was performed by high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography were as follows: The chromatographic column uses octadecylsilane bonded silica as filler; Mobile phase A: ammonium acetate buffer; Mobile phase B: methanol; The volume ratio of the mobile phase A to the mobile phase B is (70-90):(10-30).

2. The method for detecting related substances of aminophylline injection according to claim 1, characterized in that: The chromatographic column is Agilent ZORBAX Eclipse Plus C18, and its specifications are 4.6×250 mm, 5 μm.

3. The method for detecting related substances of aminophylline injection according to claim 1, characterized in that: The preparation process of the mobile phase A is as follows: Place 770.8 mg of ammonium acetate in a 1000 ml beaker and dissolve in 800 ml of water. Adjust the pH to 5.5 with glacial acetic acid and dilute with water to 1000 ml.

4. The method for detecting related substances of aminophylline injection according to claim 1, characterized in that: In the high performance liquid chromatography method, the detection wavelength is 270 nm, the flow rate is 1.0 ml / min, the column temperature is 30° C., and the injection volume is 20 μl.

5. The method for detecting related substances of aminophylline injection according to claim 1, characterized in that: In the high performance liquid chromatography, gradient elution was performed according to the following table:

6. The method for detecting related substances of aminophylline injection according to claim 5, characterized in that: In the high performance liquid chromatography, gradient elution was performed according to the following table:

7. The method for detecting related substances of aminophylline injection according to claim 1, characterized in that: The related substances are: Impurity A: 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione; Impurity B: 3-methyl-3,7-dihydro-1H-purine-2,6-dione; Impurity C: N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)formamide; Impurity D: N-methyl-5-(methylamino)-1H-imidazole-4-carboxamide hydrochloride monohydrate; Impurity E: 1,3-dimethyl-7,9-dihydro-1H-purine-2,6,8(3H)-trione; Impurity F: 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione; Impurity G: 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione; Theophylline.

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