Method for separating and detecting impurities in bumetanib injection

Abstract

The invention discloses a method for separating and detecting impurities in a bumetanib injection, which can be used for separating and detecting an impurity B and other unknown single impurities in the bumetanib injection at the same time, has a remarkable separation effect, and can be used for detecting impurities in the bumetanib injection. The content of impurities in the bumetanib injection is evaluated and calculated, and an effective and simple bumetanib injection quality control method is provided. Therefore, the method has good application value and popularization prospect.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more specifically to a method for simultaneously separating and detecting impurities in bumetanide injection. Background Technology

[0002] Bumetanide, chemical name: 5-n-butylamino-4-phenoxy-3-aminosulfonylbenzyl. Its effects on water and electrolyte excretion are essentially the same as furosemide, but its diuretic effect is 20–60 times stronger. It primarily inhibits the active reabsorption of NaCl in the thick-walled ascending limb of the loop of Henle, and also inhibits Na+ reabsorption in the proximal tubule, but has no effect on the distal tubule; therefore, its potassium excretion effect is less than that of furosemide. It can inhibit the activity of prostaglandin-degrading enzymes, increasing prostaglandin E2 levels, thereby exhibiting a vasodilatory effect. This dilation of renal vessels, reduction of renal vascular resistance, and increase in renal blood flow, especially deep cortical blood flow, are crucial to the diuretic effect of bumetanide and form the theoretical basis for its use in preventing acute renal failure. Furthermore, unlike other diuretics, loop diuretics do not decrease glomerular filtration rate while increasing renal tubular fluid flow, possibly due to reduced chloride flow through the macula densa, thus weakening or blocking the glomerular-tubular balance. Bumetanide dilates pulmonary volume veins and reduces pulmonary capillary permeability. Combined with its diuretic effect, this reduces venous return and lowers left ventricular end-diastolic pressure, which is beneficial for the treatment of acute left ventricular failure. Because bumetanide reduces pulmonary capillary permeability, it provides a theoretical basis for its use in treating adult respiratory distress syndrome.

[0003] Bumetanide injection is a well-known diuretic administered by injection. Its clinical efficacy and safety have been clinically recognized. Because it enters the human body directly, quality control of its preparation process is particularly important. Related substances (impurities) and their content are direct indicators of drug purity; controlling or reducing the quantity and content of impurities is a key aspect of drug quality research. Summary of the Invention

[0004] The present invention aims to provide a method for simultaneously separating and detecting impurities in bumetanide injection to solve the above-mentioned problems.

[0005] To achieve the above objectives, the following technical methods can be used:

[0006] One approach is as follows: The method for separating and detecting impurities in the above-mentioned bumetanide injection includes the following steps: preparing a test solution, a control solution and a system suitability solution, and using high performance liquid chromatography to detect impurities in the solution;

[0007] Chromatographic conditions include:

[0008] The chromatographic column is packed with pentafluorophenylpropylsilane-bonded silica gel or octadecylsilane-bonded silica gel;

[0009] Mobile phase A is 0.1% phosphoric acid (pH 2.9–3.1)-acetonitrile (70:30), preferably pH 3.0;

[0010] Mobile phase B is 0.1% phosphoric acid (pH 2.9–3.1)-acetonitrile (20:80), preferably pH 3.0;

[0011] Elution method: gradient elution.

[0012] Furthermore, the chromatographic column used is either Shim-pack Scepter C18 or a column with equivalent performance; preferably, Shim-pack Scepter C18 is used.

[0013] Furthermore, the specifications of the chromatographic column are 4.6 mm × 150 mm and 3 μm.

[0014] Furthermore, the elution gradients under the above chromatographic conditions are shown in the table below:

[0015] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 15 100 0 30 20 80 45 20 80 45.1 100 0

[0016] Furthermore, the detection wavelength for the above chromatographic conditions is 210–230 nm, preferably 220 nm.

[0017] Furthermore, the column temperature of the above-mentioned chromatographic column is 25℃~35℃, preferably 30℃.

[0018] Furthermore, the column flow rate of the above-mentioned chromatographic column is 0.6 to 1.0 ml / min, preferably 0.8 ml / min.

[0019] This invention provides a simple and reliable analytical method for the quality control of bumetanide injection. Attached Figure Description

[0020] Figure 1 This is a result diagram from Example 2 of the method of the present invention;

[0021] Figure 2 This is a result diagram from Example 3 of the method of the present invention;

[0022] Figure 3 This is a result diagram from Example 4 of the method of the present invention. Detailed Implementation

[0023] The present invention will be further illustrated by specific embodiments below, but these are not intended to limit the scope of protection of the present invention. Those skilled in the art can make improvements to the preparation method and the instruments used within the scope of the claims, and these improvements should also be considered within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

[0024] In this invention, unless otherwise specified, all abbreviations have the conventional meanings understood by those skilled in the art.

[0025] Example 1 Selection of chromatographic conditions 1

[0026] Chromatographic conditions:

[0027] Instrument: High Performance Liquid Chromatography

[0028] Column: Octadecylsilane-bonded silica gel as the packing material;

[0029] Mobile phase: methanol-0.1% trifluoroacetic acid solution (58:42);

[0030] Detection wavelength: 220nm;

[0031] Column temperature: 25℃

[0032] Elution method: isocratic elution.

[0033] Conclusion: The separation and detection of impurities was not effective.

[0034] Example 2 Selection of chromatographic conditions 2

[0035] Chromatographic conditions:

[0036] Instrument: High Performance Liquid Chromatography

[0037] Column: Octylsilane-bonded silica gel as the packing material;

[0038] Mobile phase: methanol-water-27.2 g / L phosphate (70:25:5), pH adjusted to 7.0 with 280 g / L potassium hydroxide solution, and 2.17 g tetrahexylammonium bromide added per L;

[0039] Detection wavelength: 254nm;

[0040] Column temperature: 25℃

[0041] Elution method: isocratic elution.

[0042] The results are as follows Figure 1 Conclusion: Before the retention time is 5 minutes, impurities aggregate, resulting in poor impurity separation and detection, and high requirements are placed on the liquid phase system.

[0043] Example 3 Selection of chromatographic conditions 3

[0044] Chromatographic conditions:

[0045] Instrument: High Performance Liquid Chromatography

[0046] Column: Octadecylsilane-bonded silica gel as packing material; Shim-pack Scepter C18, 4.6 mm × 150 mm, 3 μm

[0047] Mobile phase: 0.1% trifluoroacetic acid (adjusted to pH 3.0 with triethylamine): methanol (pH 3.0) (60:40)

[0048] Detection wavelength: 220nm;

[0049] Column temperature: 30℃;

[0050] Elution method: isocratic elution.

[0051] The results are as follows Figure 2 Conclusion: Impurities C and D did not show peaks in the mixed solution, indicating poor impurity detection capability.

[0052] Example 4 Selection of chromatographic conditions 4

[0053] Chromatographic conditions:

[0054] Instrument: High Performance Liquid Chromatography

[0055] Chromatographic column: Octadecylsilane-bonded silica gel as packing material; Shim-pack Scepter C18, 4.6 mm × 150 mm, 3 μm; Mobile phase A: 0.1% phosphoric acid: acetonitrile (pH 3.0) (70:30);

[0056] Mobile phase B: 0.1% phosphoric acid: acetonitrile (pH 3.0) (20:80);

[0057] Detection wavelength: 220nm;

[0058] Column temperature: 30℃;

[0059] Elution method: gradient elution, flow rate 0.8 ml / min.

[0060] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 15 100 0 30 20 80 45 20 80 45.1 100 0 55 100 0

[0061] The results are as follows Figure 3 Conclusion: This embodiment can separate impurities A, B, C, D and ammonium hydrolysate, and can simultaneously separate and detect impurities in bumetanide injection.

[0062] Example 5 Related Verification

[0063] The chromatographic conditions were investigated according to those in Example 4. The results are as follows:

[0064] 1. Exclusivity

[0065] Prepare blank solutions, system suitability solutions, sensitivity solutions, mixed solutions, bumetanide and various impurity localization solutions, blank solutions subjected to forced degradation, and test sample solutions that are neither subjected to forced degradation nor subjected to forced degradation.

[0066] Preparation method:

[0067]

[0068]

[0069] Preparation of 0.1% phosphoric acid solution: Measure 1 ml of phosphoric acid, dilute with water to 1000 ml, adjust the pH value to 3.0±0.05 with ammonia water, mix well, and the solution is obtained.

[0070] Reference solution: Accurately measure 1.0 ml of the test solution and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well. Accurately measure 1.0 ml of the test solution and place it in a 50 ml volumetric flask. Dilute to the mark with mobile phase A and shake well to obtain the reference solution.

[0071] Test solution: Accurately measure 5.0 ml of the test sample, place it in a 10 ml volumetric flask, dilute to the mark with mobile phase A, shake well, and the solution is prepared.

[0072] Blank excipient solution: Weigh 2.3g xylitol, 0.034g anhydrous disodium hydrogen phosphate and 0.013g sodium dihydrogen phosphate monohydrate, place them in a 50ml volumetric flask, dilute with water to the mark, shake well, measure 5.0ml, place it in a 10ml volumetric flask, dilute with mobile phase A to the mark, shake well, and the blank excipient solution is prepared.

[0073] Impurity stock solution: Weigh approximately 10 mg each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H, impurity J, and impurity K reference standards, accurately weigh them, place them in 100 ml volumetric flasks, dissolve and dilute to the mark with methanol, shake well, and the solution is ready.

[0074] Bumetanib stock solution: Weigh approximately 25 mg of bumetanib reference standard accurately, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, and the solution is prepared.

[0075] Impurity localization solution: Accurately measure 5.0 ml of each impurity stock solution and place them in 50 ml volumetric flasks. Dilute to the mark with mobile phase A and shake well. Measure 1.0 ml of each of the above solutions and place them in 20 ml volumetric flasks. Dilute to the mark with mobile phase A and shake well to obtain the solution.

[0076] Bumetanide positioning solution: Accurately measure 2.0 ml of bumetanide stock solution, place it in a 10 ml volumetric flask, dilute to the mark with mobile phase A, and shake well.

[0077] Mixed solution: Accurately measure 5.0 ml of each impurity stock solution and place it in the same 100 ml volumetric flask. Dilute to the mark with mobile phase A and shake well. Accurately measure 2.0 ml of each impurity stock solution and place it in a 20 ml volumetric flask. Accurately measure 4.0 ml of bumetanide stock solution and place it in the same 20 ml volumetric flask. Dilute to the mark with mobile phase A and shake well to obtain the mixed solution.

[0078] System suitability solution: Weigh approximately 2 mg of impurity reference standard B and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with methanol and shake well. Measure 1 ml of the solution and place it in a 10 ml volumetric flask. Weigh approximately 10 mg of bumetanide reference standard and place it in the same 10 ml volumetric flask. Dissolve and dilute to the mark with methanol and shake well. Measure 5 ml of the solution and place it in a 20 ml volumetric flask. Dilute to the mark with mobile phase A and shake well.

[0079] Take the above solutions and inject them into a high-performance liquid chromatograph according to the determined relevant substance detection method. Record the chromatograms and the detection data are shown in Table 1.

[0080] Table 1. Detection results of the specificity-impurity localization experiment.

[0081]

[0082] The results showed that under the above chromatographic conditions, the peak positions of bumetanide and all known impurities in the chromatograms of the blank solutions did not interfere with each other; the resolution between impurity B and the bumetanide peak was greater than 2.5, and the signal-to-noise ratio of the bumetanide peak was greater than 10, indicating that the detection method has good specificity.

[0083] 2. Limit of Quantification, Limit of Detection

[0084] Accurately measure impurities A, B, C, D, E, F, H, J, and K, and bumetanide stock solutions, respectively, and gradually dilute them. Inject the samples, record the chromatograms, and calculate the signal-to-noise ratio (SNR) of each impurity peak. The concentration at which the SNR of the target peak is approximately 3 is the limit of detection, and the concentration at which the SNR is approximately 10 is the limit of quantitation. The detection results are shown in Table 2.

[0085] Table 2 Summary of Limit of Detection and Limit of Quantitation Results

[0086]

[0087]

[0088] The results showed that the S / N ratio of the main peak in the detection limit solution was not less than 3; the S / N ratio of the main peak in the detection limit solution was not less than 10, which met the acceptance criteria.

[0089] 3. Accuracy

[0090] Three different concentration levels of each known impurity (ranging from 20% to 150% of the impurity limit) and three different concentration levels of an unknown impurity (using bumetanide as an alternative for investigation) were prepared for analysis. Triples of each concentration level were prepared and analyzed separately. The results are shown in Table 3.

[0091] Table 3 Summary of Accuracy Results

[0092]

[0093]

[0094] The experimental results showed that the average recovery rate of each impurity in the test solution with different concentrations ranged from 91.3% to 104.3%, and the RSD of the recovery rate was no greater than 10%, which met the acceptance criteria.

[0095] 4. Durability

[0096] (1) The detection temperature of the chromatographic column was fine-tuned to “25℃, 30℃, 35℃”. In the system suitability solution, the retention time of bumetanide varied from 32.575 to 32.945 min, the retention time of impurity B varied from 14.726 to 16.767 min, the resolution between impurity A and bumetanide was greater than 1.5, the signal-to-noise ratio of the bumetanide peak was greater than 100, and the absolute deviation of the content of each known impurity in the sample solution was not greater than 0.05%, and the absolute deviation of the total impurity content was not greater than 0.05%.

[0097] (2) The mobile phase was fine-tuned to pH values ​​of 2.9, 3.0, and 3.1. In the system suitability solution, the retention time of bumetanide ranged from 32.159 to 32.536 min, the retention time of impurity B ranged from 13.434 to 15.274 min, the resolution between impurity A and bumetanide was greater than 1.5, the signal-to-noise ratio of the bumetanide peak was greater than 100, and the absolute deviation of the content of each known impurity in the test solution was not greater than 0.05%, and the absolute deviation of the total impurity content was not greater than 0.05%.

[0098] (3) Fine-tune the flow rate in the chromatographic conditions to "0.6 ml / min, 0.8 ml / min, 1.0 ml / min". In the system suitability solution, the retention time of bumetanide varies from 32.469 to 32.853 min, the retention time of impurity B varies from 15.245 to 16.079 min, the resolution between impurity A and bumetanide is greater than 1.5, the signal-to-noise ratio of the bumetanide peak is greater than 100, and in the sample solution, the absolute deviation of the content of each known impurity is not greater than 0.05%, and the absolute deviation of the total impurity content is not greater than 0.05%.

[0099] The advantages of this invention are as follows: The method for separating and detecting impurities in bumetanide injection provided by this invention can simultaneously separate and detect impurity B and other unknown single impurities in bumetanide injection. Its separation effect is significant, enabling the assessment and calculation of the impurity content in bumetanide injection, and providing an effective and simple method for quality control of bumetanide injection. Therefore, this invention has excellent application value and promising prospects for promotion. It should be noted that the above preferred embodiments are only for illustrating the technical concept and features of this invention, and are intended to enable those skilled in the art to understand the content of this invention and implement it accordingly. They should not be used to limit the scope of protection of this invention. All equivalent changes or modifications made according to the spirit and essence of this invention should be covered within the scope of protection of this invention.

Claims

1. A method for separating and detecting impurities in bumetanide injection, characterized in that, The method includes the following steps: preparing a test solution, a control solution and a system suitability solution, and using high performance liquid chromatography to detect impurities in the solution; Chromatographic conditions include: The chromatographic column is packed with pentafluorophenylpropylsilane-bonded silica gel or octadecylsilane-bonded silica gel; Mobile phase A is 0.1% phosphoric acid (pH 2.9–3.1)-acetonitrile (70:30); Mobile phase B is 0.1% phosphoric acid (pH 2.9–3.1)-acetonitrile (20:80); Elution method: gradient elution.

2. The method for separating and detecting impurities in bumetanide injection according to claim 1, characterized in that, The chromatographic column used is a Shim-pack Scepter C18.

3. The method for separating and detecting impurities in bumetanide injection according to claim 1, characterized in that, The chromatographic column has dimensions of 4.6 mm × 150 mm and a diameter of 3 μm.

4. The method for separating and detecting impurities in bumetanide injection according to claim 1, characterized in that, The chromatographic conditions also include the following elution gradient:

5. The method for separating and detecting impurities in bumetanide injection according to claims 1 to 4, characterized in that, The detection wavelength for the chromatographic conditions is 210–230 nm.

6. The method for separating and detecting impurities in bumetanide injection according to claim 5, characterized in that, The detection wavelength for the chromatographic conditions is 220 nm.

7. The method for separating and detecting impurities in bumetanide injection according to claim 5, characterized in that, The column temperature of the chromatographic column is 25℃~35℃.

8. The method for separating and detecting impurities in bumetanide injection according to claim 7, characterized in that, The column temperature of the chromatographic column is 30°C.

9. The method for separating and detecting impurities in bumetanide injection according to claim 8, characterized in that, The column flow rate of the chromatographic column is 0.6–1.0 ml / min.

10. The method for separating and detecting impurities in bumetanide injection according to claim 9, characterized in that, The column flow rate of the chromatographic column is 0.8 ml / min.

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