Diprophylline injection detection method

Abstract

The invention relates to the field of biological medicine, and discloses a diprophylline injection detection method which comprises the following steps: preparing required instruments and reagents including a high performance liquid chromatograph, an octadecyl silane bonded silica gel chromatographic column, a diprophylline reference substance, an impurity B (theophylline) reference substance, an impurity C reference substance, an impurity D reference substance, purified water and HPLC-grade methanol; preparing a system applicability solution, a test solution and a reference solution, wherein the test solution is a sample solution of the diprophylline injection; setting high performance liquid chromatography conditions including a chromatographic column, a mobile phase, a flow rate, a column temperature, a detection wavelength and a sample injection volume; checking the applicability of the system; and carrying out sample injection and analysis, recording a chromatogram and calculating the impurity content. By adopting an optimized column temperature and flow velocity setting scheme, full separation of diprophylline and impurities thereof is ensured, the symmetry of chromatographic peaks is improved, and the tailing phenomenon is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, specifically to a detection method for diprophylline injection. Background Art

[0002] Diprophylline injection is a modified xanthine bronchodilator, mainly used to relieve airway spasms caused by asthma and chronic obstructive pulmonary disease (COPD). Compared with traditional theophylline drugs (such as aminophylline), diprophylline has better tolerance, fewer cardiovascular side effects and a longer action time, so it is widely used clinically.

[0003] However, the existing detection methods for diprophylline injection still have certain deficiencies in chromatographic separation, impurity determination and system stability. For example, some methods do not optimize the column temperature and flow rate of the chromatograph, resulting in reduced resolution, peak tailing or overlapping phenomena, thus affecting the quantitative accuracy. In addition, some detection procedures do not clearly set the system suitability inspection criteria, and it is easy to affect the analysis results due to fluctuations in chromatographic conditions, reducing the stability and reproducibility of the method. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a detection method for diprophylline injection, which solves the problems existing in the existing detection methods in chromatographic separation, impurity determination and system stability.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A detection method for diprophylline injection, the steps include:

[0006] Prepare the required instruments and reagents, including a high-performance liquid chromatograph, an octadecylsilane-bonded silica gel chromatographic column, a diprophylline reference substance, an impurity B (theophylline) reference substance, an impurity C reference substance, an impurity D reference substance, purified water, HPLC-grade methanol;

[0007] Prepare a system suitability solution, a test solution and a reference solution, and the test solution is a sample solution of diprophylline injection;

[0008] Set the high-performance liquid chromatography conditions, including the chromatographic column, mobile phase, flow rate, column temperature, detection wavelength and injection volume;

[0009] Conduct a system suitability inspection;

[0010] Inject the sample and analyze it, record the chromatogram and calculate the impurity content;

[0011] Calculate the impurity content according to the calculation formula and compare it with the quality standard to determine whether the diprophylline injection is qualified.

[0012] Preferably, the chromatographic column is a C18-bonded silica gel packed column.

[0013] Preferably, the mobile phase is a mixed solution of methanol and water in a ratio of 10:90 (v / v).

[0014] Preferably, the flow rate is 1.0 ml / min, the column temperature is 35 °C, the detection wavelength is 272 nm, and the injection volume is 10 μl.

[0015] Preferably, the preparation method of the system suitability solution includes dissolving and diluting appropriate amounts of diprophylline reference substance, impurity B (theophylline) reference substance, impurity C reference substance, and impurity D reference substance in water to obtain a mixed solution containing 1 mg of diprophylline and 2 μg of each impurity per 1 ml.

[0016] Preferably, the preparation method of the test solution includes accurately measuring 1 ml of diprophylline injection, placing it in a 25-ml volumetric flask, diluting it to the mark with water, shaking well, then accurately measuring 5 ml and placing it in a 25-ml volumetric flask, and continuing to dilute it to the mark with water and shaking well.

[0017] Preferably, the preparation method of the reference solution includes accurately weighing about 10 mg of diprophylline reference substance, placing it in a 200-ml volumetric flask, dissolving it with water and diluting it to the mark, shaking well, then accurately measuring 2 ml and placing it in a 50-ml volumetric flask, and diluting it to the mark with water and shaking well.

[0018] Preferably, the calculation formula for the impurity content is as follows:

[0019]

[0020] Where: F: The correction factors for impurity B, impurity C, and impurity D are 0.64, 1.0, and 1.0 respectively, and the correction factor for other individual impurities is 1.0;

[0021] m RS : The weighed amount of the reference substance, in mg;

[0022] P RS : The content of the reference substance, in %;

[0023] V RS : The dilution factor of the reference solution;

[0024] A RS : The average value of the main peak areas of 5 consecutive injections of the reference solution;

[0025] A U : The peak areas of each impurity in the test solution;

[0026] V U : The dilution factor of the test solution;

[0027] C: Labeled amount, unit: 125 mg / ml;

[0028] V: Volume of the test sample taken, unit: ml.

[0029] Preferably, the quality standard requirements for each impurity in the diprophylline injection are as follows: impurity B (theophylline) ≤ 0.2%, impurity C ≤ 0.2%, impurity D ≤ 0.2%, other single impurities ≤ 0.2%, total impurities ≤ 0.5%, and the ignore limit is 0.05%.

[0030] Preferably, the judgment criteria for the system suitability test include that impurity B, diprophylline, impurity C, and impurity D appear in sequence in the chromatogram, and the resolution between the diprophylline peak and the impurity C peak is greater than 2.5.

[0031] The present invention provides a detection method for diprophylline injection. It has the following beneficial effects:

[0032] By adopting an optimized column temperature and flow rate setting scheme, the present invention ensures the full separation of diprophylline and its impurities, improves the symmetry of chromatographic peaks, and reduces the tailing phenomenon. Compared with the problems of decreased resolution and increased background noise caused by unstable column temperature control or unreasonable flow rate setting in the prior art, the present invention significantly improves the peak shape and enhances the accuracy and repeatability of detection. Description of the Drawings

[0033] Figure 1 It is a schematic diagram of the steps of the present invention. Detailed Embodiments

[0034] The technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0035] The existing detection method for diprophylline injection adopts the ChP2020 standard; when reproducing the related substances method, it is found that the theophylline (impurity B) and the main component peak cannot be effectively separated; referring to the chromatographic conditions of the EP11.0 / BP2024 raw material standard and optimizing the column temperature and flow rate, the system suitability meets the requirements and can be used for the detection of related substances of this product.

[0036]

[0037]

[0038]

[0039]

[0040] In this implementation, the detection of diprophylline injection was established with reference to the current version of the EP11.0 / BP2024 method. Based on the EP11.0 / BP2024 standard method, the column temperature and flow rate were optimized for the chromatographic conditions. After methodological verification, it is applicable to the detection of this product.

[0041] Please refer to the appendix Figure 1 , the embodiment of the present invention provides a method for detecting diprophylline injection. The related substances in diprophylline injection are detected by high performance liquid chromatography (HPLC). The steps include:

[0042] Prepare the required instruments and reagents, including a high performance liquid chromatograph, an octadecylsilane bonded silica gel chromatographic column, diprophylline reference substance, impurity B (theophylline) reference substance, impurity C reference substance, impurity D reference substance, purified water, HPLC grade methanol;

[0043] Prepare the system suitability solution, test solution and reference solution. The test solution is the sample solution of diprophylline injection;

[0044]

[0045] Set the high performance liquid chromatography conditions, including the chromatographic column, mobile phase, flow rate, column temperature, detection wavelength and injection volume;

[0046]

[0047]

[0048] Conduct a system suitability test;

[0049] Inject the sample and analyze it, record the chromatogram and calculate the impurity content;

[0050] Calculate the impurity content according to the calculation formula and compare it with the quality standard to determine whether the diprophylline injection is qualified.

[0051] The calculation formula for the impurity content is:

[0052]

[0053] Where: F: The correction factors for impurity B, impurity C, and impurity D are 0.64, 1.0, and 1.0 respectively, and the correction factor for other individual impurities is 1.0;

[0054] m RS : The weighed amount of the reference substance, in mg;

[0055] P RS : The content of the reference substance, in %;

[0056] V RS : Dilution factor of the reference solution;

[0057] A RS : Average value of the main peak areas of 5 consecutive injections of the reference solution;

[0058] A U : Peak areas of each impurity in the test solution;

[0059] V U : Dilution factor of the test solution;

[0060] C: Labeled amount, in the unit of 125 mg / ml;

[0061] V: Volume of the test sample taken, in the unit of ml.

[0062] According to the "General Principles for Validation of Analytical Methods" (Part IV, 9101) in the Chinese Pharmacopoeia 2020 Edition, the developed method was verified for system suitability, specificity, linearity and range, precision (injection precision, repeatability, intermediate precision), accuracy, and robustness (including solution stability), and the results were all satisfactory, indicating that the developed method meets the requirements for the related substances test of this product. The results are summarized as follows:

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting diphylline injection, characterized in that the steps include: Prepare the necessary instruments and reagents, including high performance liquid chromatography, octadecylsilane bonded silica gel chromatography column, dihydroxypropylphylline reference substance, impurity B (theophylline) reference substance, impurity C reference substance, impurity D reference substance, purified water, and HPLC grade methanol; Prepare system suitability solution, test solution and reference solution. The test solution is the sample solution of diprophylline injection; Set HPLC conditions, including column, mobile phase, flow rate, column temperature, detection wavelength, and injection volume; Conduct system suitability checks; Perform sample injection and analysis, record chromatograms and calculate impurity levels; Calculate the impurity content according to the calculation formula and compare it with the quality standard to determine whether the diprophylline injection is qualified.

2. The method for detecting dyphylline injection according to claim 1, characterized in that: The chromatographic column is a C18 bonded silica gel filling column.

3. The method for detecting dyphylline injection according to claim 1, characterized in that: The mobile phase is a mixed solution of methanol and water in a ratio of 10:90 (v / v).

4. The method for detecting dyphylline injection according to claim 1, characterized in that: The flow rate was 1.0 ml / min, the column temperature was 35° C., the detection wavelength was 272 nm, and the injection volume was 10 μl.

5. The method for detecting dyphylline injection according to claim 1, characterized in that: The preparation method of the system suitability solution comprises adding appropriate amounts of a diphylline reference substance, an impurity B (theophylline) reference substance, an impurity C reference substance, and an impurity D reference substance to water for dissolution and dilution to prepare a mixed solution containing 1 mg of diphylline and 2 μg of each impurity in each ml.

6. The method for detecting dyphylline injection according to claim 1, characterized in that: The preparation method of the test solution comprises the following steps: accurately measuring 1 ml of diprophylline injection, placing it in a 25 ml volumetric bottle, adding water to dilute it to the mark, shaking it well, accurately measuring 5 ml again, placing it in a 25 ml volumetric bottle, continuing to add water to dilute it to the mark, and shaking it well.

7. The method for detecting dyphylline injection according to claim 1, characterized in that: The preparation method of the reference solution comprises the following steps: accurately weighing about 10 mg of the diphylline reference substance, placing it in a 200 ml volumetric bottle, adding water to dissolve and dilute to the mark, shaking well, then accurately measuring 2 ml, placing it in a 50 ml volumetric bottle, adding water to dilute to the mark, and shaking well.

8. The method for detecting dyphylline injection according to claim 1, characterized in that: The calculation formula of the impurity content is: Where: F: The correction factors of impurity B, impurity C, and impurity D are 0.64, 1.0, and 1.0 respectively, and the correction factor of other single impurities is 1.0; m RS : The sample weight of the reference substance, in mg; P RS : The content of the reference substance, in %; V RS : Dilution multiple of reference solution; A RS : The average of the main peak areas of 5 consecutive injections of the reference solution; A U : The peak area of ​​each impurity in the test solution; V U : dilution multiple of the test solution; C: labeled amount, unit is 125mg / ml; V: The volume of the test sample, in ml.

9. The method for detecting dyphylline injection according to claim 1, characterized in that: The quality standard requirements of each impurity in the dihydroxypropylphylline injection are: impurity B (theophylline) ≤ 0.2%, impurity C ≤ 0.2%, impurity D ≤ 0.2%, other single impurities ≤ 0.2%, total impurities ≤ 0.5%, and the negligible limit is 0.05%.

10. The method for detecting dyphylline injection according to claim 1, characterized in that: The judgment criteria of the system suitability check include that impurity B, diprophylline, impurity C, and impurity D appear in order in the chromatogram, and the separation degree between the diprophylline peak and the impurity C peak is greater than 2.5.

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