Analysis method of colistin sulfate component for injection and related substances

Abstract

The invention provides a method for analyzing colistin sulfate components and related substances for injection, which adopts a high performance liquid chromatography, and is characterized in that the chromatographic conditions of the high performance liquid chromatography are as follows: a mobile phase A is a sodium sulfate solution-acetonitrile mixed solution, the mass percentage content of acetonitrile is 21.2-21.8%, the mobile phase B is a mobile phase C, the mobile phase C is a mobile phase D, and the mobile phase D is a mobile phase E; and the mobile phase B is acetonitrile. The method can be simultaneously applicable to detection of related substances and components of colistin sulfate raw material medicines, colistin sulfate freeze-dried preparations for injection and the like, and is good in stability.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis, and in particular to an analysis method for colistin sulfate components for injection and related substances. Background Art

[0002] The quality standards of the Chinese Pharmacopoeia (ChP) Part II monographs do not include the variety of colistin sulfate for injection, and there are no related substance inspection items in the quality standards of colistin sulfate. The European Pharmacopoeia (EP) also does not have a quality standard for colistin sulfate for injection. The quality standards of colistin sulfate raw materials have established related substance and component inspection items. When using this method in different equipment and different laboratories, it was found that the method sensitivity and separation effect could not meet the system applicability requirements of the method, making it difficult to actually apply this method. Summary of the invention

[0003] In order to overcome the above defects, the method was applied to the detection of related substances and components of colistin sulfate bulk drug and colistin sulfate injection and its lyophilized preparations by optimizing chromatographic parameters, including selecting appropriate chromatographic column models, increasing injection volume, modifying mobile phase ratio, column temperature and flow rate, and adding gradient elution procedures. At the same time, through a series of adjustment processes, the main parameters affecting the separation effect of chromatographic peaks were studied, providing guidance for detection.

[0004] The present invention provides a method for analyzing components of colistin sulfate for injection and related substances, using high performance liquid chromatography, characterized in that:

[0005] The chromatographic conditions of the high performance liquid chromatography are as follows:

[0006] Mobile phase A is a sodium sulfate solution-acetonitrile mixture, wherein the mass percentage content of acetonitrile is 21.2-21.8%;

[0007] Mobile phase B was acetonitrile.

[0008] Furthermore, the present invention provides a method for analyzing components of colistin sulfate for injection and related substances, which is also characterized by:

[0009] The sodium sulfate solution has a mass percent concentration of 0.3-0.5% and a pH of 2.2-2.6.

[0010] Furthermore, the present invention provides a method for analyzing components of colistin sulfate for injection and related substances, which is also characterized by:

[0011] The flow rate is 0.7ml to 0.9ml per minute.

[0012] Furthermore, the present invention provides a method for analyzing components of colistin sulfate for injection and related substances, which is also characterized by:

[0013] First, perform isocratic elution with mobile phase A, and then perform linear gradient elution after impurity B is eluted.

[0014] Furthermore, the present invention provides a method for analyzing components of colistin sulfate for injection and related substances, which is also characterized by:

[0015] The linear gradient elution process is as follows: after impurity B emerges, mobile phase A is continuously reduced to 65%, while mobile phase B is continuously increased to 35%.

[0016] The specific plan is as follows:

[0017]

[0018] Furthermore, the present invention provides a method for analyzing components of colistin sulfate for injection and related substances, which is also characterized by:

[0019] A chromatographic column with a length of 250 mm and a particle size of 3 μm was selected.

[0020] Furthermore, the present invention provides a method for analyzing components of colistin sulfate for injection and related substances, which is also characterized by:

[0021] The column temperature was 38-42°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 , spectrum obtained using European Pharmacopoeia 11.0 method, Rs = 2.81;

[0023] Figure 2 , the spectrum obtained by the method of the best embodiment, Rs = 3.6;

[0024] Figure 3 , Typical chromatogram of system suitability solution;

[0025] Figure 4 , the effect of mobile phase ratio on the separation of colistin 2,3-dehydroE1 and colistin E1;

[0026] Figure 5 , the effect of flow rate on the separation degree of colistin 2,3-dehydro E1 and colistin E1-1;

[0027] Figure 6 , the effect of flow rate on the separation degree of colistin 2,3-dehydro E1 and colistin E1-2;

[0028] Figure 7 , the effect of column temperature on the separation of colistin 2,3-dehydroE1 and colistin E1;

[0029] Figure 8 , sensitivity solution spectra with different injection volumes. Specific embodiments

[0030] Example 1. Analysis method

[0031] Components: Determined by high performance liquid chromatography (General Rule 0512).

[0032] Solvent: water-acetonitrile (80:20).

[0033] Test solution: Take about 5.0 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0034] System suitability solution: Accurately weigh about 5.0 mg of colistin system suitability A reference substance, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0035] Sensitivity solution: Accurately measure an appropriate amount of system suitability solution and quantitatively dilute it with solvent to make each 1 ml contain approximately 1.75 μg of colistin E1.

[0036] Optimal chromatographic conditions

[0037] Use octadecylsilane bonded silica gel as filler (YMC-Triart C18, 250 mm × 4.6 mm, 3 μm or a chromatographic column with equivalent performance);

[0038] Sodium sulfate solution-acetonitrile was used as mobile phase A.

[0039] Regarding the preparation of sodium sulfate solution: dissolve 4.46 g of anhydrous sodium sulfate in 900 ml of water, adjust the pH value to 2.2-2.6 (most preferably 2.4) with phosphoric acid, and then dilute with water to 1000 ml;

[0040] Sodium sulfate solution: acetonitrile = 78.5: 21.5 (optimal ratio), the applicable range of acetonitrile can be selected from 21.2%-21.8%;

[0041] Acetonitrile was the mobile phase B;

[0042] First, perform isocratic elution with mobile phase A, and after impurity B is eluted (about 50 minutes), immediately perform linear gradient elution according to the table below; the flow rate is 0.7-0.9 ml per minute (optimal 0.8 ml), the column temperature is 38-42°C (optimal 40°C); the detection wavelength is 215 nm; the injection volume is 100 μl.

[0043]

[0044] Note: After the above gradient elution program is run, balance at the initial ratio for about 15 minutes (it can be adjusted according to actual conditions, and the balance time can be increased or decreased, mainly depending on the length of the flow path of the HPLC equipment. The flow path length varies for different brands of equipment and different installation methods).

[0045] System suitability requirements: In the system suitability solution chromatogram, the retention time of the colistin E1 peak is about 40 minutes. The relative retention times of individual components and individual impurities are shown in the table below. The separation between the colistin E6 peak and the colistin E2-I peak should be no less than 2.0; the separation between the colistin 2,3-dehydrogenated E1 peak and the colistin E1 peak should be no less than 3.0; the peak-to-valley ratio of impurity A to colistin E2 should be no less than 1.1. In the sensitivity solution chromatogram, the signal-to-noise ratio of the colistin E1 peak should be no less than 10.

[0046]

[0047]

[0048] Determination method: Accurately measure the test solution, inject it into the liquid chromatograph, and record the chromatogram.

[0049] Component calculation: The content of colistin E1-I, colistin E3, colistin E1-7MOA, colistin E6, colistin E1-Nva, colistin E4 and E2-Val, colistin E2-I, the content of colistin 2,3-dehydrogenated E1 after multiplying the peak area by the correction factor 0.3, and the total content of colistin E1, E2, E3, E4, E6, E1-7MOA, E1-I, E1-Nva, E2-I, E2-Val and 2,3-dehydrogenated E1 components were calculated by area normalization method. Peaks smaller than the main peak area of ​​the sensitivity solution were ignored.

[0050] Related substances: Determined by high performance liquid chromatography (General Rule 0512).

[0051] Test solution, system suitability solution, sensitivity solution, chromatographic conditions, system suitability and assay methods are listed under the components.

[0052] Calculation of related substances: Calculate impurity A, impurity B, other individual impurities and the total amount of impurities by area normalization method. Peaks smaller than the main peak area of ​​the sensitivity solution are ignored.

[0053] Example 2. Condition screening example

[0054] 2.1. Screening of chromatographic columns

[0055] Several chromatographic columns of different brands, models and specifications were tested using the EP method, and it was found that the chromatographic column that best met the EP system suitability requirements (in the system suitability solution chromatogram, the separation between the colistin E6 peak and the colistin E2-I peak should be no less than 2.0; the separation between the colistin 2,3-dehydrogenated E1 peak and the colistin E1 peak should be no less than 3.0; the peak-to-valley ratio of impurity A to colistin E2 should be no less than 1.1) was the YMC-Triart C18 (250×4.6mm, 3μm) column. The data are shown in Table 1, and the values ​​that do not meet the requirements have a darker background color.

[0056] The test found that the separation of colistin 2,3-dehydrogen E1 and colistin E1 is the key point of this method and the main problem to be solved. The separation of the chromatographic column with a particle size of 3μm is better than that of 5μm, and the column length of 250mm has a better separation effect than that of 150mm.

[0057] Table 1 Column screening system suitability solution results

[0058]

[0059] 2.2. Mobile phase ratio study

[0060] The effect of the ratio of organic phase to aqueous phase in the mobile phase on the separation degree (Rs) of colistin 2,3-dehydro E1 and colistin E1 was investigated. Mobile phase A was aqueous phase and mobile phase B was organic phase. By slightly increasing or decreasing the ratio of organic phase, the results showed that the retention time and separation degree of the target peak would be significantly affected. The results are as follows Figure 4 As shown:

[0061] First, a slight change in the proportion of organic phase in the mobile phase (1%) has a very significant effect on the retention time (>5min); compared with improving the separation degree, this influencing factor is more important for maintaining the stability of the retention time of each component, so the ratio of aqueous phase to organic phase needs to be strictly controlled.

[0062] Secondly, as the retention time of the target peak changes, the separation degree also shows a certain change pattern. The separation degree will increase to a certain extent as the retention time is extended; however, compared with the rate of change of the retention time, the improvement in separation degree is not very obvious.

[0063] Finally, due to slight differences in proportional valves between different instruments, there is a greater risk in maintaining the stability of the retention time of each component. Especially for isocratic elution methods, it is recommended to run the two phases in a single channel.

[0064] 2.3. Flow rate study

[0065] The study of flow rate found that reducing the flow rate can improve the separation of target peaks, such as Figure 5As shown. And for small particle size chromatographic columns with high column pressure, appropriately reducing the flow rate and column pressure can extend the service life of the chromatographic column.

[0066] Using a YMC-Triart C18 (250×4.6mm, 3μm) column, the effect of flow rate was investigated again based on the adjusted mobile phase ratio (A:B=78.5%:21.5%). The results are shown in Figure 6 , at a flow rate of less than 1.0 ml / min, the target peak separation can meet the requirements.

[0067] 2.4. Selection of column temperature

[0068] The column temperature was investigated and it was found that a change in column temperature within a small range, such as 50°C ± 2°C, had no effect on the retention time and separation of the target peak, but if the temperature changed by ±5°C to ±10°C, it would affect the separation of the target peak. Figure 7 As shown, When the column temperature is within the range of 40℃~50℃, the separation degree is less affected by the change of column temperature. When the temperature is higher than 50℃, the column temperature The higher the value, the smaller the separation, and the chromatographic column is prone to damage after long-term use. . Less than 40℃ When the column temperature is low, the peak is slower and the testing speed of the sample is too slow. Therefore, it is not an ideal method.

[0069] The column temperature of a general chromatographic column is recommended to be in the range of 20℃~40℃. The upper pressure limit of a 250mm chromatographic column of YMC-Triart C18 is 25MPa. The column temperature of 40℃~50℃ and the column flow rate of 0.8ml / min~1.0ml / min can be selected as appropriate parameters.

[0070] 2.5. Others

[0071] The ion concentration, pH value, injection volume, diluent, and elution mode of the mobile phase in the EP method were also studied.

[0072] Increasing the ion concentration to 50 mM and changing the pH value by ±0.1 had little effect on the resolution of the target peaks.

[0073] Changing the diluent to water has little effect on sample dissolution and peak shape.

[0074] Due to the influence of different detectors and baselines on detection sensitivity, the applicability of the method on different devices was improved by increasing the injection volume from 20 μl to 100 μl. The results are shown in Table 2.

[0075] The same batch of samples were tested in isocratic elution mode, and 6 test sample solutions (SPL) were prepared in parallel. The number of unknown impurities detected in each sample was different, and the amount of the same unknown impurity detected was inconsistent. Therefore, the isocratic elution method cannot completely elute the strongly retained impurities or the existing residues, and the repeatability of the test samples does not meet the requirements. The results are shown in Table 3.

[0076] On the basis of isocratic elution of the mobile phase, a linear gradient elution program is further added, so that strongly retained impurities on the chromatographic column can be separated and detected, while improving the precision of the method detection results.

[0077] Table 2 Results of sensitivity solution investigation with different injection volumes

[0078]

[0079]

[0080] Table 3 Results of sample repeatability investigation of isocratic elution method

[0081]

[0082] Example 3. Verification Experiment Example

[0083] The optimized method can be used to test conventional retained samples and forced degradation samples. The results are shown in Table 4. The method can effectively separate and detect degradation impurities and ensure the quality conservation of the test results.

[0084] The repeatability of the method was investigated using the same batch of samples, and the results are shown in Table 5. The repeatability of the method was good. Other methodological validation items have been investigated, and the method has been verified to be suitable for the detection of colistin sulfate for injection and its raw materials.

[0085] Table 4 Test results of different samples

[0086]

[0087]

[0088] Table 5 Repeatability test results of the same sample

[0089]

[0090] In summary, the establishment of the above method is used to detect the components and related substances of colistin sulfate for injection and its raw materials, which provides a method for the study of key quality attributes of the product and at the same time safeguards the quality control of the product.

Claims

1. A method for analyzing components and related substances of colistin sulfate for injection, using high performance liquid chromatography, characterized in that: The chromatographic conditions of the HPLC are as follows: Mobile phase A is a sodium sulfate solution-acetonitrile mixture, wherein the mass percentage content of acetonitrile is 21.2-21.8%; Mobile phase B was acetonitrile.

2. The method for analyzing the components and related substances of colistin sulfate for injection according to claim 1, characterized in that: The sodium sulfate solution has a mass percent concentration of 0.3-0.5% and a pH of 2.2-2.

6.

3. The method for analyzing the components and related substances of colistin sulfate for injection according to claim 1, characterized in that: The flow rate is 0.7ml to 0.9ml per minute.

4. The method for analyzing the components and related substances of colistin sulfate for injection according to claim 1, characterized in that: First, perform isocratic elution with mobile phase A, and then perform linear gradient elution after impurity B is eluted.

5. The method for analyzing the components and related substances of colistin sulfate for injection according to claim 4, characterized in that: The linear gradient elution process is as follows: after impurity B emerges, mobile phase A is continuously reduced to 65%, while mobile phase B is continuously increased to 35%.

6. The method for analyzing the components and related substances of colistin sulfate for injection according to claim 1, characterized in that: A chromatographic column with a length of 250 mm and a particle size of 3 μm was selected.

7. The method for analyzing the components and related substances of colistin sulfate for injection according to claim 6, characterized in that: The column temperature was 38-42°C.

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