Method for determining impurity I in bumetanib injection

Abstract

The invention relates to the technical field of drug analysis, and discloses a method for determining impurity I in bumetanib injection, which comprises the following steps: using a reverse high performance liquid chromatograph and an ultraviolet detector, taking ACE Excel C18-PFP as a chromatographic column, taking 0.1% phosphoric acid aqueous solution as a mobile phase A, taking methanol-acetonitrile (40: 60) as a mobile phase B, adopting a gradient elution procedure, and determining the impurity I in the bumetanib injection according to the detection wavelength of 220nm, the sample injection volume of 10mu L and the flow velocity of 1.0 mL / min. The column temperature is 30 DEG C; the content of the impurity I in the bumetanib injection can be accurately detected, the blank of no impurity I detection method report is filled, and method verification is carried out. The method designed by the invention can be used for separating and accurately determining the impurity I in the bumetanib injection, and is high in specificity, high in sensitivity and good in precision and accuracy; and the quality control of the bumetanib injection is effectively enhanced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and to a method for determining impurity I in bumetanide injection, specifically a method for detecting degradation impurity I in bumetanide injection using liquid chromatography. Background Technology

[0002] Bumetanide is a loop diuretic primarily used to treat edema and hypertension caused by heart failure, cirrhosis, or kidney disease. Its chemical name is 3-butylamino-4-phenoxy-5-aminosulfonylbenzoic acid, and its molecular formula is C2. 17 H 20 N2O5S, with a molecular weight of 364.42, has the following structural formula: Bumetanib is mainly formulated as an injection. Bumetanib injection is available in various strengths, including: (1) 1 mL: 0.5 mg, (2) 2 mL: 0.5 mg, (3) 2 mL: 1 mg, (4) 4 mL: 1 mg, and (5) 4 mL: 2 mg. Bumetanib injection (4 mL: 2 mg) is a sterile aqueous solution of xylitol and phosphate. In its stability study, it was found that a new impurity I was generated during storage. The generation of impurity I will affect its quality. Therefore, it is necessary to study and control impurity I in bumetanib injection (4 mL: 2 mg) to ensure the safety of the product quality.

[0003] The structure of impurity I in bumetanide injection was determined (see [link]). Figure 1 and Figure 2 Its chemical name is 3-(butanoamino)-5-(N-(2-hydroxyacetyl)aminosulfonyl)-4-phenoxybenzoic acid, and its chemical formula is C. 19 H 22 N₂O₇S, with a molecular weight of 422.45, has the following structural formula: Impurity I Currently, no reports have been found regarding the detection method for impurity I in bumetanide injection. To better control the quality of bumetanide injection (4mL:2mg), a method for determining impurity I in bumetanide injection is provided. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to fill the gap in methods for detecting impurity I in bumetanide injection by providing a method for separating and determining impurity I in bumetanide injection using high-performance liquid chromatography (HPLC), thereby achieving quality control and ensuring the safety of bumetanide injection. This invention can accurately determine impurity I in bumetanide injection, and the method has been validated. The method designed in this invention exhibits high specificity, high sensitivity, and good precision and accuracy in determining impurity I in bumetanide injection.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for determining impurity I in bumetanide injection, wherein the chemical name of bumetanide is 3-butano-4-phenoxy-5-aminosulfonylbenzoic acid, and its structural formula is as follows: Impurity I is chemically named 3-(butanoamino)-5-(N-(2-hydroxyacetyl)aminosulfonyl)-4-phenoxybenzoic acid, and its structural formula is as follows: The method includes the following steps: S1 Preparation of reference solution: Take appropriate amounts of impurity I and bumetanide reference standard respectively, accurately weigh them, dissolve them in acetonitrile and dilute them with solvent to prepare a mixed solution containing 1 μg of degradation impurity I and bumetanide per 1 mL; S2 Preparation of the test solution: Take bumetanide injection as the test solution; S3 detection: Take equal amounts of the test solution and the reference solution and inject them into the liquid chromatograph for determination; Chromatographic conditions include: Reverse liquid chromatography was used, with acidic solution as mobile phase A and a mixture of methanol and acetonitrile as mobile phase B for gradient elution.

[0006] The bumetanide injection solution is available in a 4mL:2mg specification.

[0007] Preferably, the modifier in the mobile phase A is one of formic acid, acetic acid, phosphoric acid or trifluoroacetic acid; the modifier is preferably phosphoric acid, with a concentration of 0.05% to 0.3%, more preferably 0.1%.

[0008] Preferably, the volume ratio of methanol to acetonitrile in the mobile phase B is 20:80 to 80:20; more preferably, it is 40:60.

[0009] Preferably, the chromatographic column is one of C18, C18-AR, C18-PFP or a column with equivalent performance, preferably C18-PFP; more preferably ACE Excel C18-PFP, with specifications of 4.6mm×250nm and 5μm.

[0010] Preferably, the column temperature of the chromatographic column is 25~40℃, and more preferably 30℃.

[0011] Preferably, the detector is an ultraviolet detector with a detection wavelength of 210~250nm, preferably 220nm.

[0012] Preferably, the injection volume is 5~50μL, and more preferably 10μL.

[0013] Preferably, the flow rate of the mobile phase is 0.9 mL / min to 1.1 mL / min, and more preferably 1.0 mL / min.

[0014] Preferably, the gradient elution procedure is as follows: At 0 min, the volume ratio of mobile phase A was 63-67%, and the volume ratio of mobile phase B was 33-37%. For 8 minutes, the volume ratio of mobile phase A was 41-45%, and the volume ratio of mobile phase B was 55-59%. For 15 minutes, the volume ratio of mobile phase A was 41-45%, and the volume ratio of mobile phase B was 55-59%. For 30 minutes, the volume ratio of mobile phase A was 32-37%, and the volume ratio of mobile phase B was 63-68%. For 40 minutes, the volume ratio of mobile phase A was 28-32%, and the volume ratio of mobile phase B was 68-72%. For 42 minutes, the volume ratio of mobile phase A was 63-67%, and the volume ratio of mobile phase B was 33-37%. For 50 minutes, the volume ratio of mobile phase A was 63-67%, and the volume ratio of mobile phase B was 33-37%. The gradient elution procedure is preferably: At 0 min, the volume ratio of mobile phase A was 65%, and the volume ratio of mobile phase B was 35%. After 8 minutes, the volume ratio of mobile phase A was 43%, and the volume ratio of mobile phase B was 57%. After 15 minutes, the volume ratio of mobile phase A was 43%, and the volume ratio of mobile phase B was 57%. For 30 minutes, the volume ratio of mobile phase A was 35%, and the volume ratio of mobile phase B was 65%. For 40 minutes, the volume ratio of mobile phase A was 30%, and the volume ratio of mobile phase B was 70%. After 42 minutes, the volume ratio of mobile phase A was 65%, and the volume ratio of mobile phase B was 35%. For 50 minutes, the volume ratio of mobile phase A was 65% and the volume ratio of mobile phase B was 35%.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. The detection method of the present invention fills the gap in the current lack of detection of impurity I in bumetanide injection.

[0016] 2. The detection method of the present invention uses high performance liquid chromatography to separate impurity I, which can accurately determine the content of impurity I in bumetanide injection, and the method has been validated.

[0017] 3. The detection method of the present invention has strong specificity, high sensitivity, good precision and accuracy, which can enhance the quality control of bumetanide injection, especially the bumetanide injection with a specification of 4mL:2mg, and is of great significance to the quality controllability and safety of bumetanide injection. Attached Figure Description

[0018] Figure 1 Mass spectrometry for degrading impurity I.

[0019] Figure 2 To degrade impurity I 1 H-NMR spectrum.

[0020] Figure 3 This is a liquid chromatogram of the blank solution in Example 1 of this disclosure.

[0021] Figure 4 This is a liquid chromatogram of the reference solution in Example 1 of this disclosure.

[0022] Figure 5 This is a liquid chromatogram of the test solution in Example 1 of this disclosure.

[0023] Figure 6 This is a liquid chromatogram of the limit-of-quantity solution in Example 1 of this disclosure.

[0024] Figure 7 for Figure 6 A magnified view of the liquid chromatogram of a solution with the medium limit of quantitation.

[0025] Figure 8 This is a liquid chromatogram of the detection limit solution in Example 1 of this disclosure.

[0026] Figure 9 for Figure 8 A magnified view of the liquid chromatogram of the solution with the detection limit in the middle.

[0027] Figure 10This is a liquid chromatogram of the reference solution in Example 2 of this disclosure.

[0028] Figure 11 This is a liquid chromatogram of the test solution in Example 2 of this disclosure.

[0029] Figure 12 This is a liquid chromatogram of the reference solution in Example 3 of this disclosure.

[0030] Figure 13 This is a liquid chromatogram of the test solution in Example 3 of this disclosure. Detailed Implementation

[0031] The sources of impurity I reference standard and the test sample used in the embodiments of this disclosure are as follows: Impurity I reference standard was obtained from Pharmace ChemI Lab, batch number 25-BUM-93-47, with a purity of 97.02%. The sample to be tested came from Chengdu Lier Pharmaceutical Co., Ltd., with batch number 230703.

[0032] Furthermore, the instruments and other reagents used in the embodiments of this disclosure are all commercially available.

[0033] Example 1 A method for determining impurity I in bumetanide injection includes the following steps: S1 Preparation of reference solution: Take appropriate amounts of impurity I and bumetanide reference standard respectively, accurately weigh them, dissolve them in acetonitrile, dilute to the mark with solvent, mix well, and prepare a mixed solution containing 1 μg of impurity I and bumetanide per 1 mL; the solvent is water-acetonitrile solution (water to acetonitrile volume ratio is 40:60). S2 Preparation of test solution: Take bumetanide injection with a specification of 4mL:2mg as the test solution; S3 detection: Take equal amounts of the test solution and the reference solution and inject them into a high-performance liquid chromatograph for determination; The chromatographic conditions are as follows: Column: ACE Excel C18-PFP, 4.6 mm × 250 mm, 5 μm; Detection wavelength: 220nm; Flow rate: 1.0 mL / min; Column temperature: 30℃; Mobile phase: 0.1% mol / L phosphoric acid was used as mobile phase A, and methanol-acetonitrile volume ratio of 40:60 was used as mobile phase B; Injection volume: 10 μL; The elution procedure is shown in Table 1: Table 1 Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 8 43 57 15 43 57 30 35 65 40 30 70 42 65 35 50 65 35 Inject the solvent, reference solution, and test solution into the liquid chromatograph under the chromatographic conditions described above, and record the chromatograms, as follows: Figures 3-9 As shown in the figure; the data results of Example 1 are shown in Table 2: Table 2 Therefore, under the above chromatographic conditions, the solvent peak does not interfere with impurity I and bumetanide, and the minimum resolution between impurity I peak and adjacent impurity peak is 2.22.

[0034] Example 2 A method for determining impurity I in bumetanide injection includes the following steps: S1 Preparation of reference solution: Take appropriate amounts of impurity I and bumetanide reference standard respectively, accurately weigh them, dissolve them in acetonitrile, dilute to the mark with solvent, mix well, and prepare a mixed solution containing 1 μg of impurity I and bumetanide per 1 mL; the solvent is water-acetonitrile solution (water to acetonitrile volume ratio is 40:60). S2 Preparation of test solution: Take bumetanide injection with a specification of 4mL:2mg as the test solution; S3 detection: Take equal amounts of the test solution and the reference solution and inject them into a high-performance liquid chromatograph for determination; The chromatographic conditions are as follows: Column: ACE Excel C18-PFP, 4.6 mm × 250 mm, 5 μm; Detection wavelength: 220nm; Flow rate: 0.9 mL / min; Column temperature: 28℃; Mobile phase: 0.1% mol / L phosphoric acid was used as mobile phase A, and methanol-acetonitrile volume ratio of 40:60 was used as mobile phase B; Injection volume: 10 μL; The elution procedure is shown in Table 3: Table 3 Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 8 43 57 15 43 57 30 35 65 40 30 70 42 65 35 50 65 35 Inject the solvent, reference solution, and test solution into the liquid chromatograph under the chromatographic conditions described above, and record the chromatograms, as follows: Figures 10-11 As shown in the figure; the data results of Example 2 are shown in Table 4: Table 4 Therefore, under the above chromatographic conditions, the solvent peak does not interfere with impurity I and bumetanide, and the minimum resolution between impurity I peak and adjacent impurity peak is 1.57.

[0035] Example 3 A method for determining impurity I in bumetanide injection includes the following steps: S1 Preparation of reference solution: Take appropriate amounts of impurity I and bumetanide reference standard respectively, accurately weigh them, dissolve them in acetonitrile, dilute to the mark with solvent, mix well, and prepare a mixed solution containing 1 μg of impurity I and bumetanide per 1 mL; the solvent is water-acetonitrile solution (water to acetonitrile volume ratio is 40:60). S2 Preparation of test solution: Take bumetanide injection with a specification of 4mL:2mg as the test solution; S3 detection: Take equal amounts of the test solution and the reference solution and inject them into the high performance liquid chromatograph for determination; The chromatographic conditions are as follows: Column: ACE Excel C18-PFP, 4.6 mm × 250 mm, 5 μm; Detection wavelength: 220nm; Flow rate: 1.1 mL / min; Column temperature: 32℃; Mobile phase: 0.1% mol / L phosphoric acid was used as mobile phase A, and methanol-acetonitrile volume ratio of 40:60 was used as mobile phase B; Injection volume: 10 μL; The elution procedure is shown in Table 5: Table 5 Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 8 43 57 15 43 57 30 35 65 40 30 70 42 65 35 50 65 35 Inject the solvent, reference solution, and test solution into the liquid chromatograph under the chromatographic conditions described above, and record the chromatograms, as follows: Figures 12-13 As shown; the data results of Example 3 are shown in Table 6: Table 6 Therefore, under the above chromatographic conditions, the solvent peak does not interfere with impurity I and bumetanide, and the minimum resolution between impurity I peak and adjacent impurity peak is 1.69.

[0036] To verify the effectiveness of the method provided by this invention, the specificity, sensitivity, linearity and range, accuracy and precision of the above method were further verified.

[0037] I. Specificity Test Solvent: Water-acetonitrile (40:60).

[0038] Impurity I stock solution: Accurately weigh 5 mg of impurity I reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, shake well, accurately measure 1 mL, place it in a 50 mL volumetric flask, dilute to the mark with solvent, shake well, and prepare a solution containing 10 μg of impurity I per mL.

[0039] Bumetanib stock solution: Accurately weigh 5 mg of bumetanib reference standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with acetonitrile, shake well, accurately measure 1 mL, place it in a 50 mL volumetric flask, dilute to the mark with solvent, shake well, and prepare a solution containing 10 μg bumetanib per mL.

[0040] Reference solution: Accurately measure 1 mL each of impurity I stock solution and bumetanide stock solution, place them in a 10 mL volumetric flask, dilute to the mark with solvent, shake well, and prepare a mixed solution containing 1 μg each of impurity I and bumetanide per 1 mL.

[0041] Test solution: Take the test sample, and each 1 mL contains 0.5 mg of bumetanide.

[0042] Spiked test solution: Take 1 mL of impurity I stock solution and place it in a 10 mL volumetric flask. Dilute to the mark with the test solution and shake well (the amount of impurity I added is 100% of the relative limit (0.2%)).

[0043] Take the solvent, reference solution, test solution and spiked test solution, and inject them into the chromatographic conditions of Example 1 for analysis.

[0044] The results showed that the solvent did not interfere with impurity I and bumetanide. The resolution of impurity I and bumetanide in the reference solution, as well as the resolution of impurity I peak and adjacent impurity peak in the test solution and the spiked (relative limit 100%) test solution, were all not less than 1.5.

[0045] II. Sensitivity Test Solvent: Water-acetonitrile (40:60).

[0046] Take appropriate amounts of impurity I stock solution and bumetanide stock solution, and dilute them stepwise with solvent to prepare limit of concentration (SRC) solution (SRC ≥ 10) and limit of detection (SRC ≥ 3); the sensitivity test results are shown in Table 7. Table 7 Serial Number Detection limit (μg / mL) Equivalent to the concentration ratio of the test sample solution (%) Detection limit signal-to-noise ratio Limit of quantitation (μg / mL) Equivalent to the concentration ratio of the test sample solution (%) Quantitative limit signal-to-noise ratio Impurity I 0.013 0.003 8.14 0.044 0.009 17.25 Bumetani 0.013 0.003 7.30 0.042 0.008 19.11 The results show that the method has high response sensitivity to impurity I and bumetanide.

[0047] III. Linearity and Range Test Bumetanide stock solution and impurity I stock solution were prepared into linear solutions at the limit of quantitation and the equivalent limit concentration (1 μg / mL) of 10%, 20%, 50%, 100%, and 200%, respectively. Solutions of each concentration were analyzed according to the chromatographic conditions of Example 1, from lowest to highest concentration. The linearity test results are shown in Table 8. Table 8 name Linear range (μg / mL) Linear equations linear correlation coefficient Impurity I 0.044~2.182 y = 29.4382x + 0.1055 1.0000 Bumetani 0.042~2.110 y = 33.6398x + 0.1721 1.0000 The results showed that the linear relationship of impurity I was good in the concentration range of 0.044~2.182 μg / mL (equivalent to 4.4%~218.2% of the limit), and the linear relationship of bumetanide was good in the concentration range of 0.042~2.110 μg / mL (equivalent to 4.2%~211.0% of the limit).

[0048] IV. Precision Test Six test solutions were prepared in parallel by the same analyst according to the test solution preparation method under "Specificity Test". They were analyzed by injection under the chromatographic conditions of Example 1. The content and RSD value of impurity I were calculated using the external standard method. The results of precision verification are shown in Table 9. Table 9 Serial Number 1 2 3 4 5 6 mean RSD Impurity I content (%) 0.078 0.080 0.079 0.080 0.085 0.085 0.081 3.8 V. Accuracy Test The accuracy of the method was assessed by adding impurity I stock solution to the test solution and determining the recovery rate. Following the specificity test method, two aliquots of the test solution and six aliquots of the spiked test solution were prepared in parallel and analyzed under the chromatographic conditions of Example 1. The recovery rate was calculated by dividing the difference between the measured value of impurity I in the spiked test solution and the measured value of impurity I in the test solution by the amount of impurity I added. The results are shown in Table 10. Table 10 Serial Number 1 2 3 4 5 6 mean RSD Impurity I recovery rate (%) 106.68 105.58 107.04 105.05 106.15 105.34 106.0 0.75 The results showed that the recovery rate of impurity I was between 105.05% and 107.04%, with an average of 106.0% and an RSD of 0.75%. The method had good accuracy and could accurately detect the content of impurity I.

[0049] Therefore, the present invention discloses a method for detecting impurity I in bumetanide injection using liquid chromatography, which can determine the content of impurity I, thereby providing data support for the screening of bumetanide injection preparation process and quality control, and is of great significance in terms of product quality controllability and safety.

[0050] The above description is only a specific embodiment of the present invention, but the technical features of the present invention are not limited thereto. Any changes or modifications made by those skilled in the art within the scope of the present invention are covered by the patent scope of the present invention.

Claims

1. A method for the determination of Impurity I of a bumetanide injection solution, characterized in that, The impurity I is 3-(butylamino)-5-(N-(2-hydroxyacetyl)sulfamoyl)-4-phenoxybenzoic acid, and its structural formula is as follows: The method comprises the following steps: S1: preparing a control solution: taking impurity I and bumetanide control substance respectively, precisely weighing, dissolving in acetonitrile and diluting with solvent to prepare a mixed solution containing 1 μg of impurity I and bumetanide in 1 mL; S2: preparing a test solution: taking bumetanide injection as the test solution; S3: detection: taking equal amounts of the test solution and the control solution respectively, and injecting into a liquid chromatograph for determination; The chromatographic conditions comprise: An acid solution is used as mobile phase A, and methanol and acetonitrile are mixed to prepare mobile phase B for gradient elution.

2. The method for determining the impurity I of the bumetanide injection according to claim 1, characterized in that, The specification of the bumetanide injection is 4 mL: 2 mg.

3. The method of claim 1, wherein the impurity I is: ###0001### 3 The modifier in the mobile phase A is one of formic acid, acetic acid, phosphoric acid or trifluoroacetic acid; preferably, the modifier is phosphoric acid, and the concentration thereof is 0.05%-0.3%, more preferably 0.1%.

4. The method of claim 1, wherein the impurity I is: ###0002### The volume ratio of methanol to acetonitrile in the mobile phase B is 20:80-80:20, preferably 40:

60.

5. The method of claim 1, wherein the impurity I is: ###0002### The chromatographic column is one of C18, C18-AR and C18-PFP, preferably C18-PFP; more preferably, the chromatographic column is ACE Excel C18-PFP, with a specification of 4.6 mm x 250 nm and 5 μm.

6. The method of claim 1, wherein the impurity I is: ###0002### The column temperature of the chromatographic column is 25-40°C, preferably 30°C.

7. The method of claim 1, wherein the impurity I is: ###0002### The detector is an ultraviolet detector, and the detection wavelength is 210-250 nm, preferably 220 nm.

8. The method of claim 1, wherein the impurity I is: ###0002### The injection volume is 5-50 μL, preferably 10 μL.

9. The method of claim 1, wherein the impurity I is: ###0002### The flow rate of the mobile phase is 0.9 mL / min-1.1 mL / min, preferably 1.0 mL / min.

10. A method of determining the presence of Impurity I in a bumetanide injection solution according to any one of claims 1 to 9, wherein, The gradient elution program is as follows: 0 min, the volume ratio of mobile phase A is 63-67%, and the volume ratio of mobile phase B is 33-37%; 8 min, the volume ratio of mobile phase A is 41-45%, and the volume ratio of mobile phase B is 55-59%; 15 min, the volume ratio of mobile phase A is 41-45%, and the volume ratio of mobile phase B is 55-59%; 30 min, the volume ratio of mobile phase A is 32-37%, and the volume ratio of mobile phase B is 63-68%; 40 min, the volume ratio of mobile phase A is 28-32%, and the volume ratio of mobile phase B is 68-72%; 42 min, the volume ratio of mobile phase A is 63-67%, and the volume ratio of mobile phase B is 33-37%; 50 min, the volume ratio of mobile phase A is 63-67%, and the volume ratio of mobile phase B is 33-37%; Preferably, the gradient elution program is as follows: 0 min, the volume ratio of mobile phase A is 65%, and the volume ratio of mobile phase B is 35%; 8 min, the volume ratio of mobile phase A is 43%, and the volume ratio of mobile phase B is 57%; 15 min, the volume ratio of mobile phase A is 43%, and the volume ratio of mobile phase B is 57%; 30 min, the volume ratio of mobile phase A is 35%, and the volume ratio of mobile phase B is 65%. 40 min, mobile phase A 30%, mobile phase B 70%; 42 min, mobile phase A 65%, mobile phase B 35%; 50 min, mobile phase A 65%, mobile phase B 35%.

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