Method for detecting related impurities in aminophylline injection

Abstract

The invention discloses a method for detecting related impurities in aminophylline injection, which belongs to the technical field of pharmaceutical analysis, and comprises the following steps: (1) preparing a better mobile phase system for high performance liquid chromatography; (2) setting a gradient elution program and instrument parameters of high performance liquid chromatography; (3) sample preparation: preparing a system applicability solution, a test solution and a contrast solution; and (4) a determination method: precisely measuring the system applicability solution, the test solution and the contrast solution, respectively injecting into a liquid chromatograph, and recording chromatograms. The method for detecting related impurities in aminophylline injection has the advantages of convenient inspection operation, high detection specificity, good detection capability, high detection sensitivity, low detection limit / quantitation limit, good selectivity for detection instruments and chromatographic columns, high recovery rate, good durability, high detection stability and accuracy, and wide application prospect. The detection method can accurately reflect the real content of related impurities in the aminophylline injection, so that the quality control of the aminophylline injection is improved, and the quality and the use safety of medicines are ensured.

Description

Technical Field

[0001] The present invention relates to an optimized and improved high performance liquid chromatography detection method, in particular to a detection method for related impurities in aminophylline injection, belonging to the technical field of pharmaceutical analysis. Technical Background

[0002] Aminophylline is a double salt of theophylline and diethylamine. Its pharmacological effect mainly comes from theophylline, and ethylenediamine enhances its water solubility. The relaxation effect of aminophylline on bronchial smooth muscle is the strongest. It can dilate the bronchi, increase the vital capacity, and has a relatively long-lasting effect, especially significant for spasmodic bronchi. In addition, aminophylline also has the effects of dilating the coronary artery, increasing myocardial blood supply, and strengthening cardiac contractility.

[0003] Aminophylline was first marketed in the United States in 1940 in tablet form, and the listed company was GD SEARLE LLC. In 1982, aminophylline injection was approved for marketing in the United States, and the listed company was HOSPIRA, with specifications of 250mg / 10ml and 500mg / 20ml. In 1950, aminophylline injection was approved for marketing in Japan, and the listed company was Eisai Co., Ltd. Aminophylline has four dosage forms in China, namely tablets, capsules, injections, and oral liquids, with tablets being the most common.

[0004] According to the quality standard of aminophylline included in the second part of the Chinese Pharmacopoeia 2020 edition, its chemical name is 1,3 - dimethyl - 3,7 - dihydro - 1H - purine - 2,6 - dione - 1,2 - ethylenediamine salt dihydrate or anhydrous substance, and its chemical structural formula is as follows:

[0005]

[0006] Aminophylline injection is included in the Chinese Pharmacopoeia (ChP2020), the United States Pharmacopoeia (USP - NF2022), the Japanese Pharmacopoeia (JP18), the British Pharmacopoeia (BP2023), etc. Only the United States Pharmacopoeia (USP - NF2022) lists specific impurities under the related substances item, which is impurity D. By referring to the United States Pharmacopoeia (USP - NF2022) and combining the production process route of aminophylline raw materials and the degradation pathway of the raw drug, the impurity profile of aminophylline is analyzed. It can be seen that there may be 9 organic compound impurities in aminophylline, and the specific impurity structures are as follows:

[0007]

[0008] In the prior art, the Chinese Pharmacopoeia 2020 edition, the British Pharmacopoeia 2023, and the United States Pharmacopoeia-NF2022 all contain methods for testing impurities related to aminophylline and aminophylline injection. (1) The method for detecting impurities related to aminophylline injection included in ChP2020 is: high performance liquid chromatography in isocratic elution mode, with a detection wavelength of 271nm, a C18 chromatographic column, and no column temperature requirement. After reproducing the method included in the Chinese Pharmacopoeia 2020, it was found that impurity H overlapped with impurity C, and the method specificity failed to meet the requirements. (2) The method for detecting impurities related to aminophylline injection included in the United States Pharmacopoeia is ultra-high performance liquid chromatography in gradient elution mode. Ultra-high performance liquid chromatography and equipment are not very popular, and after reproducing this method, it was found that the separation degree between impurity I and impurity D was less than 1.5, and baseline separation could not be achieved, and the method specificity failed to meet the requirements. (3) The impurity detection method for aminophylline injection listed in the British Pharmacopoeia is: high performance liquid chromatography in isocratic elution mode, with a detection wavelength of 272 nm and a C18 chromatographic column. The method was reproduced and it was found that impurity H overlapped with impurity C, and the method specificity did not meet the requirements.

[0009] Therefore, it is an urgent technical problem to be solved in this technical field to develop a detection method for relevant impurities in aminophylline injection, which has convenient inspection operation, high detection specificity, good detection ability, high detection sensitivity and detection limit, good selectivity for detection instruments and chromatographic columns, good recovery rate, high durability, high detection stability and accuracy, and the detection method can accurately reflect the true content of relevant substance impurities in aminophylline injection, thereby improving the quality control of aminophylline injection and ensuring the quality and safety of drugs. Summary of the invention

[0010] The invention aims to develop a detection method for relevant impurities in aminophylline injection, which has the advantages of convenient inspection operation, high detection specificity, good detection ability, high detection sensitivity and detection limit, good selectivity for detection instruments and chromatographic columns, good recovery rate, high durability, high detection stability and accuracy. The detection method can accurately reflect the real content of relevant substance impurities in aminophylline injection, thereby improving the quality control of aminophylline injection and ensuring the quality safety of drugs.

[0011] To realize the above invention, the specific implementation scheme is as follows:

[0012] A method for detecting relevant impurities in aminophylline injection comprises the following steps:

[0013] (1) HPLC conditions

[0014] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0015] Mobile phase: Acetate buffer was used as mobile phase A, and acetate buffer-acetonitrile [(94:6)~(91:9)] was used as mobile phase B;

[0016] Detection wavelength: The detection wavelength is 269-275nm;

[0017] Flow rate: 0.7ml / min-1.3ml / min;

[0018] Column temperature: 23℃-37℃;

[0019] Perform gradient elution;

[0020] (2) Preparation of system suitability solution: Take appropriate amount of theobromine reference substance and theophylline reference substance, dissolve in solvent and dilute to make a solution containing 10 μg of each per 1 ml;

[0021] (3) Sample preparation method:

[0022] Blank solvent: mobile phase B;

[0023] Test solution: Take an appropriate amount of this product and dilute it with solvent to make a solution containing 2.5 mg of aminophylline per 1 ml;

[0024] Control solution: Accurately measure 2 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; accurately measure 5 ml, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, and shake well;

[0025] (4) Determination method: Accurately measure 20 μl of blank solvent, system suitability solution, test solution, and control solution, respectively, and inject them into the liquid chromatograph and record the chromatogram.

[0026] Preferably, in step (1), the acetate buffer in the mobile phase A is prepared as follows: 1.02 g to 1.50 g of sodium acetate is taken, 1000 ml of water is added to dissolve it, 5 ml of glacial acetic acid is added, and the mixture is mixed.

[0027] Preferably, in step (1), the acetate buffer in the mobile phase B is prepared as follows: 1.36 g of sodium acetate is taken, 1000 ml of water is added to dissolve it, 5 ml of glacial acetic acid is added, and the mixture is mixed.

[0028] Preferably, in step (1), the detection wavelength is 271 nm;

[0029] Preferably, in step (1), the column temperature is 29°C to 31°C;

[0030] Preferably, in step (1), the flow rate is: 0.9 ml / min to 1.1 ml / min;

[0031] Preferably, in step (1), the procedure of the gradient elution is as follows:

[0032]

[0033] Preferably, in step (4), in the chromatogram of the system suitability solution, the separation between all known impurities and between impurities and the main peak is greater than 1.5, and the theoretical plate number, calculated based on theophylline peak, is not less than 5000.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] 1. Use high performance liquid chromatography with a more popular gradient elution mode to replace the original ultra-high performance liquid chromatography with a gradient elution mode; the instrument cost of high performance liquid chromatography is lower, the detection operation is simpler and easier, and the sample preparation and pre-treatment requirements can be reduced, which can realize large-scale detection of multiple samples, thereby reducing the detection cost of the product.

[0036] 2. The detection ability of the optimized and improved detection method is better. The separation between each impurity and between the impurity and the main peak can achieve effective baseline separation, and the separation is greater than 1.5. The problem of impurity C and impurity H peaks overlapping and unable to be separated in the prior art has been improved. A photodiode array detector (PDA) was used to investigate the degradation products of this product under acid, alkali, high temperature, oxidation, light and other conditions. In the chromatograms of the test sample solutions in each destruction test, the separation between the theophylline peak and the adjacent impurity peak was greater than 1.5, the peak purity angle was less than the peak purity threshold, and the purity of the theophylline peak met the requirements.

[0037] 3. The methodological validation was completed, and the specificity and system applicability met the requirements; the detection limit and quantification limit of each impurity and the main peak of aminophylline met the requirements; the peak area and concentration of theophylline and each impurity showed a good linear relationship in the range of quantification limit to 200% of the limit concentration, and the good linear correlation coefficient was greater than 0.990, and the ratio of the Y-axis intercept to the 100% concentration response value was within 10%, which met the validation requirements; the impurity external standard method and the self-control method with correction factor were used to calculate: the recovery rate of each impurity at the quantitative limit concentration was between 70% and 130%, and the recovery rate at the limit concentration of 100% and 150% was between 80% and 120%, and RSD≤10%, which met the validation requirements. Repeatability test 6 sample solutions, the detection amount of each impurity was greater than 0.05% and the RSD value was less than 15%, which met the validation requirements.

[0038] Compared with the prior art, the complete analytical method verification more scientifically and objectively proves that the analytical detection method of the present invention can effectively, accurately and stably detect the content of each impurity of the relevant substance, which is beneficial to improving the safety of clinical medication and provides a variety of selected analytical detection methods for the relevant substances in the aminophylline injection in the art.

[0039] The present invention is further described below through the accompanying drawings and specific embodiments, but it is not intended to limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Reproduce the spectra for the Chinese Pharmacopoeia 2020 test method;

[0041] Figure 2 Reproduce spectra for the USP-NF test method;

[0042] Figure 3 Reproduce spectra for the British Pharmacopoeia-NF test method;

[0043] Figure 4 The spectrum of the blank solvent in the detection method of relevant impurities in the aminophylline injection in Example 1 of the present invention;

[0044] Figure 5 The spectrum of the system suitability solution in the detection method of relevant impurities in the aminophylline injection in Example 1 of the present invention;

[0045] Figure 6 The figure is the spectrum of the test solution in the method for detecting relevant impurities in the aminophylline injection in Example 1 of the present invention. DETAILED DESCRIPTION

[0046] Unless otherwise specified, the raw materials, additives or equipment used in the embodiments of the present invention are all commercially available products, and their models are conventional models in the art; the testing methods used are all conventional methods in the art.

[0047] Example 1

[0048] A method for detecting relevant impurities in aminophylline injection comprises the following steps:

[0049] (1) HPLC conditions

[0050] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0051] Mobile phase: acetate buffer as mobile phase A, acetate buffer-acetonitrile 94:6 as mobile phase B;

[0052] Detection wavelength: The detection wavelength is 271nm;

[0053] Flow rate: 1.0ml / min;

[0054] Column temperature: 30°C;

[0055] Perform gradient elution;

[0056] (2) Preparation of system suitability solution: Take appropriate amount of theobromine reference substance and theophylline reference substance, dissolve in solvent and dilute to make a solution containing 10 μg of each per 1 ml;

[0057] (3) Sample preparation:

[0058] Blank solvent: mobile phase B;

[0059] Test solution: Take an appropriate amount of this product and dilute it with solvent to make a solution containing 2.5 mg of aminophylline per 1 ml;

[0060] Control solution: Accurately measure 2 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; accurately measure 5 ml, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, and shake well;

[0061] (4) Determination method: Accurately measure blank solvent, system suitability solution, test solution, and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram;

[0062] In step (1), the acetate buffer in the mobile phase A is prepared as follows: 1.25 g of sodium acetate is added to 1000 ml of water to dissolve, and 5 ml of glacial acetic acid is added to mix well;

[0063] In step (1), the acetate buffer in the mobile phase B is prepared as follows: 1.36 g of sodium acetate is added to 1000 ml of water to dissolve, and 5 ml of glacial acetic acid is added to mix well;

[0064] In step (1), the procedure of gradient elution is as follows:

[0065]

[0066] In step (4), in the chromatogram of the system suitability solution, the separation between all known impurities and between impurities and the main peak is greater than 1.5, and the theoretical plate number is calculated based on theophylline peak and is not less than 5000.

[0067] like Figure 1 As shown, this is a reproduction spectrum of the detection method of the Chinese Pharmacopoeia 2020 edition (the chromatographic conditions are the same as the method included in the Chinese Pharmacopoeia); impurity H and impurity C overlap under the relevant impurity detection method of aminophylline injection included in ChP2020, and the method specificity fails to meet the requirements;

[0068] like Figure 2 As shown, it is the re-appearance spectrum of the United States Pharmacopoeia-NF detection method (the chromatographic conditions are the same as those of the method included in the United States Pharmacopoeia); the detection method for the relevant impurities of aminophylline injection included in the United States Pharmacopoeia is that the separation degree of the impurity I and the impurity D is less than 1.5, and the baseline separation cannot be achieved, and the method specificity fails to meet the requirements;

[0069] like Figure 3 As shown, the British Pharmacopoeia-NF test method reproduces the spectrum (the chromatographic conditions are the same as the method included in the British Pharmacopoeia); the impurity H and impurity C overlap under the relevant impurity detection method of aminophylline injection included in the British Pharmacopoeia, and the method specificity fails to meet the requirements;

[0070] like Figure 4 As shown, it is a spectrum of blank solvent in the detection method of relevant impurities in aminophylline injection in Example 1 of the present invention; under the chromatographic conditions of Example 1 of the present invention, the blank solvent does not interfere with the detection;

[0071] like Figure 5 As shown, it is a spectrum of the system suitability solution in the detection method of relevant impurities in the aminophylline injection in Example 1 of the present invention; under the chromatographic conditions of Example 1 of the present invention, the separation between all known impurities and between impurities and the main peak is greater than 1.5, and the impurities can be effectively separated; the theoretical plate number is calculated based on theophylline peak and is not less than 5000, and the chromatographic conditions are suitable;

[0072] like Figure 6 As shown, it is the spectrum of the test solution in the method for detecting relevant impurities in aminophylline injection in Example 1 of the present invention; the chromatographic conditions in Example 1 of the present invention can be used for detecting relevant impurities in aminophylline injection.

[0073] Example 2

[0074] A method for detecting relevant impurities in aminophylline injection comprises the following steps:

[0075] (1) HPLC conditions

[0076] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0077] Mobile phase: acetate buffer as mobile phase A, acetate buffer-acetonitrile 94:6 as mobile phase B;

[0078] Detection wavelength: The detection wavelength is 269nm;

[0079] Flow rate: 0.7ml / min;

[0080] Column temperature: 23°C;

[0081] Perform gradient elution;

[0082] (2) Preparation of system suitability solution: Take appropriate amount of theobromine reference substance and theophylline reference substance, dissolve in solvent and dilute to make a solution containing 10 μg of each per 1 ml;

[0083] (3) Sample preparation:

[0084] Blank solvent: mobile phase B;

[0085] Test solution: Take an appropriate amount of this product and dilute it with solvent to make a solution containing 2.5 mg of aminophylline per 1 ml;

[0086] Control solution: Accurately measure 2 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; accurately measure 5 ml, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, and shake well;

[0087] (5) Determination method: Accurately measure blank solvent, system suitability solution, test solution, and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram;

[0088] In step (1), the acetate buffer in the mobile phase A is prepared as follows: 1.02 g of sodium acetate is added to 1000 ml of water to dissolve, and 5 ml of glacial acetic acid is added to mix well;

[0089] In step (1), the acetate buffer in the mobile phase B is prepared as follows: 1.36 g of sodium acetate is added to 1000 ml of water to dissolve, and 5 ml of glacial acetic acid is added to mix well;

[0090] In step (1), the procedure of gradient elution is as follows:

[0091]

[0092] In step (4), in the chromatogram of the system suitability solution, the separation between all known impurities and between impurities and the main peak is greater than 1.5, and the theoretical plate number is calculated based on theophylline peak and is not less than 5000.

[0093] Example 3

[0094] A method for detecting relevant impurities in aminophylline injection comprises the following steps:

[0095] (1) HPLC conditions

[0096] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0097] Mobile phase: acetate buffer as mobile phase A, acetate buffer-acetonitrile 91:9 as mobile phase B;

[0098] Detection wavelength: The detection wavelength is 275nm;

[0099] Flow rate: 1.3 ml / min;

[0100] Column temperature: 37°C;

[0101] Perform gradient elution;

[0102] (2) System suitability solution: Take appropriate amounts of theobromine reference substance and theophylline reference substance, dissolve in solvent and dilute to make a solution containing 10 μg of each per 1 ml;

[0103] (3) Sample preparation:

[0104] Blank solvent: mobile phase B;

[0105] Test solution: Take an appropriate amount of this product and dilute it with solvent to make a solution containing about 2.5 mg of aminophylline per 1 ml;

[0106] Control solution: Accurately measure 2 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; accurately measure 5 ml, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, and shake well;

[0107] (4) Determination method: Accurately measure blank solvent, system suitability solution, test solution, and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram;

[0108] In step (1), the acetate buffer in the mobile phase A is prepared as follows: 1.50 g of sodium acetate is added to 1000 ml of water to dissolve it, and 5 ml of glacial acetic acid is added to mix well;

[0109] In step (1), the acetate buffer in the mobile phase B is prepared as follows: 1.36 g of sodium acetate is added to 1000 ml of water to dissolve, and 5 ml of glacial acetic acid is added to mix well;

[0110] In step (1), the procedure of gradient elution is as follows:

[0111]

[0112] In step (2), in the chromatogram of the system suitability solution, the separation between all known impurities and between impurities and the main peak is greater than 1.5, and the theoretical plate number is calculated based on theophylline peak and is not less than 5000.

[0113] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting relevant impurities in aminophylline injection, comprising the following steps: (1) HPLC conditions Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase: Acetate buffer was used as mobile phase A, and acetate buffer-acetonitrile [(94:6)~(91:9)] was used as mobile phase B; Detection wavelength: The detection wavelength is 269-275nm; Flow rate: 0.7ml / min-1.3ml / min; Column temperature: 23℃-37℃; Perform gradient elution; (2) Preparation of system suitability solution: Take appropriate amount of theobromine reference substance and theophylline reference substance, dissolve in solvent and dilute to make a solution containing 10 μg of each per 1 ml; (3) Sample preparation: Blank solvent: mobile phase B; Test solution: Take an appropriate amount of this product and dilute it with solvent to make a solution containing 2.5 mg of aminophylline per 1 ml; Control solution: Measure 2 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; accurately measure 5 ml, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, and shake well; (4) Determination method: Take blank solvent, system suitability solution, test solution and control solution and inject them into liquid chromatograph respectively, and record the chromatogram.

2. The method for detecting relevant impurities in the aminophylline injection according to claim 1, characterized in that: In step (1), the ratio of acetate buffer to acetonitrile in the mobile phase B is (94:6) to (91:9).

3. The method for detecting relevant impurities in aminophylline injection according to claim 2, characterized in that: In step (1), the acetate buffer in the mobile phase B is prepared as follows: 1.36 g of sodium acetate is added to 1000 ml of water to dissolve it, and 5 ml of glacial acetic acid is added to mix well.

4. The method for detecting relevant impurities in aminophylline injection according to claim 2, characterized in that: In step (1), the acetate buffer in mobile phase A is prepared as follows: 1.02 g to 1.50 g of sodium acetate is added to 1000 ml of water to dissolve it, and 5 ml of glacial acetic acid is added to mix well.

5. The method for detecting relevant impurities in aminophylline injection according to claim 4, characterized in that: In step (1), the procedure of gradient elution is as follows:

6. The method for detecting relevant impurities in aminophylline injection according to claim 5, characterized in that: In step (1), the detection wavelength is 271 nm.

7. The method for detecting relevant impurities in aminophylline injection according to claim 6, characterized in that: In step (1), the column temperature is 29°C to 31°C.

8. The method for detecting relevant impurities in aminophylline injection according to claim 7, characterized in that: In step (1), the flow rate is 0.9 ml / min-1.1 ml / min.

9. The method for detecting relevant impurities in aminophylline injection according to claim 8, characterized in that: In step (4), in the chromatogram of the system suitability solution, the separation between all known impurities and between impurities and the main peak is greater than 1.5, and the theoretical plate number is calculated based on theophylline peak and is not less than 5000.

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