Method for separating and determining furosemide and impurities thereof

Abstract

A method for separating and determining furosemide and impurities thereof comprises the following steps: 1) taking furosemide, and adding a diluent to dissolve the furosemide to obtain a sample solution with the concentration of 1mg / ml; (2) diluting the sample solution obtained in the step (1) by 100 times with a diluent to obtain a contrast solution; 3) using octadecylsilane chemically bonded silica as a chromatographic column of a filler, setting the flow velocity of a mobile phase to be 0.8-1.2 ml / min, the mobile phase being composed of a mobile phase A and a mobile phase B, the mobile phase A being a glacial acetic acid aqueous solution, the mobile phase B being tetrahydrofuran, and the mobile phases entering the chromatographic column in a gradient elution mode; and (4) respectively injecting the sample solution in the step (1) and the contrast solution in the step (2) with the same volume into a high performance liquid chromatograph according to the sample injection amount of 20 microliters, detecting by using the wavelength of 238 nm, recording a chromatogram, and completing the separation and determination of the impurities in the sample solution. The method is simple and convenient to operate, the furosemide and impurities thereof can be effectively separated and determined, and the quality of a furosemide product is favorably controlled.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and in particular to a method for separating and determining furosemide and its impurities. Background Art

[0002] Furosemide is a diuretic mainly used to treat edema, hypertension and heart failure. Its mechanism of action is to inhibit the reabsorption of sodium and chloride by the renal tubules, increase the amount of excretion in urine, and reduce blood volume and cardiac load. Its molecular formula is C 12 H 11 ClN2O5S, the structural formula is shown in formula (a).

[0003] At present, the standards of furosemide are included in the Chinese Pharmacopoeia, British Pharmacopoeia, European Pharmacopoeia and United States Pharmacopoeia. When testing with reference to the existing European Pharmacopoeia standards, it was found that the impurity detection under the chromatographic conditions was incomplete; when testing with reference to the Chinese Pharmacopoeia standards, it was found that the impurity separation under the chromatographic conditions was poor.

[0004] The chemical name of furosemide is 2-[(2-furanylmethyl)amino]-5-(sulfamoyl)-4-chlorobenzoic acid, and its structural formula is shown in formula (a). In the process of synthesizing this compound, key materials and unknown impurities may affect the purity and quality of the drug due to incomplete removal. These key materials and unknown impurities and the resulting degradation products are what are commonly referred to as related substances (i.e., impurities) in drug quality control. There are nine known impurities that are mainly controlled in the synthesis of furosemide, namely: EP impurity B, EP impurity A, EP impurity C, EP impurity D, EP impurity E, and EP impurity F, and their structural formulas are shown in formulas (b), (c), (d), (e), (f), (g), and (h), respectively.

[0005]

[0006]

[0007] It can be seen that furosemide has many impurities and similar structures, which makes separation difficult. In addition, the polarity of each impurity is different. On the premise of separating furosemide from each impurity, the separation between impurities must also be met, making detection difficult. It is difficult to achieve effective separation of furosemide and impurities and between impurities using conventional detection methods, resulting in difficulty in controlling the quality of furosemide.

[0008] In order to accurately control the quality of furosemide, it is necessary to develop a method that can simply, quickly and accurately separate and detect related substances in furosemide. Summary of the invention

[0009] The purpose of the present invention is to provide a method for separating and determining furosemide and its impurities in view of the deficiencies in the prior art. The method is simple and convenient to operate, can effectively separate and determine furosemide and its impurities, and is conducive to controlling the quality of furosemide products.

[0010] The technical solution of the present invention is: a method for separating and determining furosemide and its impurities, comprising the following steps:

[0011] 1) Prepare sample solution

[0012] Take furosemide or a preparation containing furosemide, add a diluent to dissolve it, and obtain a sample solution with a concentration of 1 mg / ml;

[0013] 2) Prepare control solution

[0014] Take the sample solution obtained in step 1), add a diluent and dilute it 100 times to obtain a control solution;

[0015] 3) The chromatographic column is set with a mobile phase flow rate of 0.8-1.2 ml / min, wherein the mobile phase is composed of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of glacial acetic acid, and mobile phase B is tetrahydrofuran. The mobile phase enters the chromatographic column in a gradient elution mode. At 0 minutes, the volume percentage of mobile phase A is 92-88%, and the volume percentage of mobile phase B is 8-12%; from 0 minutes to 5 minutes, the volume percentage of mobile phase A decreases linearly to 88-80%, and the volume percentage of mobile phase B increases linearly to 8-20%; from 5 minutes to 18 minutes, the volume percentage of mobile phase A decreases linearly to 80-70%, and the volume percentage of mobile phase B increases linearly to 20-30%; from 18 minutes to 40 minutes, mobile phase A increases linearly to 10-20%. The volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%; from 40 minutes to 48 minutes, the volume percentage of mobile phase A decreases linearly to 70-60%, and the volume percentage of mobile phase B increases linearly to 30-40%; from 48 minutes to 52 minutes, the volume percentage of mobile phase A increases linearly to 60-80%, and the volume percentage of mobile phase B decreases linearly to 40-20%; from 52 minutes to 55 minutes, the volume percentage of mobile phase A increases linearly to 80-90%, and the volume percentage of mobile phase B decreases linearly to 20-10%; from 55 minutes to 65 minutes, the volume percentage of mobile phase A increases linearly to 92-88%, and the volume percentage of mobile phase B decreases linearly to 8%-12%;

[0016] 4) Injecting equal volumes of the sample solution of step 1) and the control solution of step 2) into a high performance liquid chromatograph, respectively, using a wavelength of 236 to 240 nm for detection, recording a chromatogram, and completing the separation and determination of impurities in the sample solution.

[0017] Furthermore, the diluent in step 1) and step 2) is a mixture of glacial acetic acid and acetonitrile aqueous solution, and the volume ratio of glacial acetic acid to acetonitrile aqueous solution is 11:489.

[0018] Preferably, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:1.

[0019] Furthermore, in step 3), the glacial acetic acid aqueous solution is a mixture of glacial acetic acid and water, and the volume ratio of glacial acetic acid to water is 1:70.

[0020] Preferably, in step 3), the mobile phase enters the chromatographic column by gradient elution, where at 0 minute, the volume percentage of mobile phase A is 90%, and the volume percentage of mobile phase B is 10%; from 0 minute to 5 minute, the volume percentage of mobile phase A linearly decreases to 80%, and the volume percentage of mobile phase B linearly increases to 20%; from 5 minute to 18 minute, the volume percentage of mobile phase A linearly decreases to 70%, and the volume percentage of mobile phase B linearly increases to 30%; from 18 minute to 40 minute, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%; from 40 minutes to 48 minutes, the volume percentage of mobile phase A linearly decreases to 60%, and the volume percentage of mobile phase B linearly increases to 40%; from 48 minutes to 52 minutes, the volume percentage of mobile phase A linearly increases to 80%, and the volume percentage of mobile phase B linearly decreases to 20%; from 52 minutes to 55 minutes, the volume percentage of mobile phase A linearly increases to 90%, and the volume percentage of mobile phase B linearly decreases to 10%; from 55 minutes to 65 minutes, the volume percentage of mobile phase A is 90%, and the volume percentage of mobile phase B is 10%;

[0021] Furthermore, the chromatographic column in step 3) uses octadecylsilane bonded silica gel as a filler or a chromatographic column with equivalent performance, and the chromatographic column model is ZORBAX SB-C18, with a specification of 4.6 mm×250 mm, 5 μm.

[0022] Preferably, the flow rate of the mobile phase in step 3) is 1 ml / min, and the injection volume in step 4) is 20 μl.

[0023] Preferably, step 4) is detected at a wavelength of 238 nm, and the column temperature of the chromatographic column is 30-40°C.

[0024] Preferably, the column temperature of the chromatographic column is 35°C.

[0025] The above technical solution has the following beneficial effects:

[0026] 1. The separation and determination method of the present invention uses a chromatographic column with octadecylsilane bonded silica gel as a filler, an aqueous glacial acetic acid solution as a mobile phase A, and tetrahydrofuran as a mobile phase B, which can effectively separate and detect furosemide and its impurities. A mixture of glacial acetic acid and acetonitrile is selected as a diluent to dissolve the sample, eliminating the interference of the solvent peak and the solvent effect. The volume ratio of mobile phase A and mobile phase B is adjusted by gradient elution to ensure that furosemide and impurities, and impurities and impurities can be effectively separated. If the volume ratio of mobile phase A and mobile phase B is not adjusted by gradient, the main peak and the impurity peak with similar polarity will have poor separation or even cannot be separated.

[0027] 2. The separation and determination method of the present invention adopts the self-control method with correction factor to calculate the content of each impurity in the test sample, which has high accuracy and the minimum relative detection limit of each impurity is 0.005%, indicating that impurities greater than 0.005% can be detected.

[0028] 3. The mobile phase A and mobile phase B used in the separation and determination method of the present invention are simple and convenient to prepare, and the reagents used are all common reagents, which are economical and affordable. According to the polarity of each component, the mobile phase gradient is adjusted to effectively separate furosemide from adjacent impurity peaks, degradation products, starting materials, and impurities of various known structures, accurately determine impurities, and the peak shape is symmetrical, the column efficiency is high, and the method running time is suitable, thereby solving the problem that furosemide (impurities: including starting materials, degradation products) is difficult to separate and determine, and the quality of furosemide can be effectively guaranteed.

[0029] Further description is given below in conjunction with the accompanying drawings and specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The liquid chromatogram of the mixture of glacial acetic acid, acetonitrile and water in Example 1;

[0031] Figure 2 is the liquid chromatogram of the mixed control solution of Example 1;

[0032] Figure 3 It is the liquid chromatogram of the sample solution of Example 2;

[0033] Figure 4 It is the liquid chromatogram of the reference solution of Example 2. DETAILED DESCRIPTION

[0034] Instruments and conditions

[0035] The high performance liquid chromatograph used was an Agilent 1260 liquid chromatograph and a Shimadzu network workstation, which were set to auto-injection. Octadecylsilane bonded silica gel was used as a filler (ZORBAX SB-C18 4.6mm×250mm, 5μm) as a separation column. The UV detector wavelength was 238nm. Mobile phase: glacial acetic acid aqueous solution was used as mobile phase A, tetrahydrofuran was used as mobile phase B, and glacial acetic acid aqueous solution was a mixture of glacial acetic acid and water, with a volume ratio of glacial acetic acid to water of 1:70. Gradient elution mode: 0 minutes, the volume percentage of mobile phase A is 90%, and the volume percentage of mobile phase B is 10%; from 0 minutes to 5 minutes, the volume percentage of mobile phase A decreases linearly to 80%, and the volume percentage of mobile phase B increases linearly to 20%; from 5 minutes to 18 minutes, the volume percentage of mobile phase A decreases linearly to 70%, and the volume percentage of mobile phase B increases linearly to 30%; from 18 minutes to 40 minutes, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%; from 40 minutes to At 48 minutes, the volume percentage of mobile phase A linearly decreased to 60%, and the volume percentage of mobile phase B linearly increased to 40%; from 48 minutes to 52 minutes, the volume percentage of mobile phase A linearly increased to 80%, and the volume percentage of mobile phase B linearly decreased to 20%; from 52 minutes to 55 minutes, the volume percentage of mobile phase A linearly increased to 90%, and the volume percentage of mobile phase B linearly decreased to 10%; from 55 minutes to 65 minutes, the volume percentage of mobile phase A was 90%, and the volume percentage of mobile phase B was 10%. The column temperature was 35°C, and the flow rate was 1.0 ml / min. The injection volume was 20 μl.

[0036] Preparation of diluent: Take equal proportions of acetonitrile and water, mix well to obtain an acetonitrile aqueous solution, add glacial acetic acid and mix well. The volume ratio of the acetonitrile aqueous solution to the glacial acetic acid is 489:11.

[0037] Example 1

[0038] Protect from light, take about 10 mg of each reference substance of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F (provided by the Technology Research and Development Branch of Chongqing Sansheng Industrial Co., Ltd.), accurately weigh, place in 100 ml volumetric flasks, add diluent to dissolve and quantitatively dilute to make a solution containing about 100 μg per 1 ml as impurity stock solution;

[0039] Protect from light, accurately measure 1.0 ml of each stock solution of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F, place in a 50 ml volumetric flask, dilute to the mark with diluent to make a solution containing about 2 μg of each impurity per 1 ml, shake well, and use as the reference solution;

[0040] Take 10 mg of furosemide, weigh it accurately, put it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, accurately measure 0.5 ml to a 50 ml volumetric flask, then accurately add 1.0 ml of each impurity stock solution, add diluent to dilute to the scale, shake well, as a mixed control solution. In the solute of the mixed control solution, the concentration of furosemide is 10 μg / ml, and the concentration of each impurity is 2 μg / ml.

[0041] Take the diluent and mixed control solution respectively, perform liquid chromatography analysis according to the above chromatographic conditions, and record the chromatogram. The results are as follows: Figure 1 , Figure 2 shown.

[0042] Figure 1 It shows that the diluent and chromatographic system do not interfere with the determination.

[0043] Figure 2 The peaks appearing in order are impurity C, impurity B, impurity F, furosemide, impurity A, impurity E, and impurity D. Figure 2 It is shown that the separation and determination method of the present invention can effectively separate impurities of unknown structure and impurities of known structure that may exist in furosemide, and can be used for the determination of furosemide impurities.

[0044] Example 2 Determination of impurities in furosemide raw material (provided by Chongqing Sansheng Industrial Co., Ltd. Technology Research and Development Branch)

[0045] Protect from light, take 10 mg of furosemide raw material, accurately weigh, place in a 10 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, as the sample solution; accurately measure 1.0 ml, place in a 100 ml volumetric flask, add diluent to dilute to scale, shake well, as the control solution. Perform liquid chromatography analysis according to the chromatographic conditions of Example 1. Record the chromatogram, the results are as follows Figure 3 , Figure 4 The impurity content in the test sample was calculated by the principal component self-comparison method with the correction factor, and the test results are shown in Table 1:

[0046] The test results of Example 2 are shown in Table 1.

[0047] Table 1

[0048]

[0049]

Claims

1. A method for separating and determining furosemide and its impurities, characterized in that: The following steps are involved: 1) Prepare sample solution Take furosemide or a preparation containing furosemide, add a diluent to dissolve it, and obtain a sample solution with a concentration of 1 mg / ml; 2) Prepare control solution Take the sample solution obtained in step 1), add a diluent and dilute it 100 times to obtain a control solution; 3) The chromatographic column is set with a mobile phase flow rate of 0.8-1.2 ml / min, wherein the mobile phase is composed of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of glacial acetic acid, and mobile phase B is tetrahydrofuran. The mobile phase enters the chromatographic column in a gradient elution mode. At 0 minutes, the volume percentage of mobile phase A is 92-88%, and the volume percentage of mobile phase B is 8-12%; from 0 minutes to 5 minutes, the volume percentage of mobile phase A decreases linearly to 88-80%, and the volume percentage of mobile phase B increases linearly to 8-20%; from 5 minutes to 18 minutes, the volume percentage of mobile phase A decreases linearly to 80-70%, and the volume percentage of mobile phase B increases linearly to 20-30%; from 18 minutes to 40 minutes, mobile phase A increases linearly to 10-20%. The volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30%; from 40 minutes to 48 minutes, the volume percentage of mobile phase A decreases linearly to 70-60%, and the volume percentage of mobile phase B increases linearly to 30-40%; from 48 minutes to 52 minutes, the volume percentage of mobile phase A increases linearly to 60-80%, and the volume percentage of mobile phase B decreases linearly to 40-20%; from 52 minutes to 55 minutes, the volume percentage of mobile phase A increases linearly to 80-90%, and the volume percentage of mobile phase B decreases linearly to 20-10%; from 55 minutes to 65 minutes, the volume percentage of mobile phase A increases linearly to 92-88%, and the volume percentage of mobile phase B decreases linearly to 8%-12%; 4) Injecting equal volumes of the sample solution of step 1) and the control solution of step 2) into a high performance liquid chromatograph, respectively, using a wavelength of 236 to 240 nm for detection, recording a chromatogram, and completing the separation and determination of impurities in the sample solution.

2. The method according to claim 1, characterized in that In step 1) and step 2), the diluent is a mixture of glacial acetic acid and acetonitrile aqueous solution, and the volume ratio of glacial acetic acid to acetonitrile aqueous solution is 11:

489.

3. The method according to claim 2, characterized in that The volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:

1.

4. The method according to claim 1, characterized in that: In step 3), the glacial acetic acid aqueous solution is a mixture of glacial acetic acid and water, and the volume ratio of glacial acetic acid to water is 1:

70.

5. The method according to claim 1, characterized in that The flow rate of the mobile phase in step 3) was 1 ml / min, and the injection volume in step 4) was 20 μl.

6. The method according to claim 1, characterized in that Step 3) The mobile phase enters the chromatographic column in a gradient elution mode, where at 0 minute, the volume percentage of mobile phase A is 90%, and the volume percentage of mobile phase B is 10%; from 0 minute to 5 minute, the volume percentage of mobile phase A linearly decreases to 80%, and the volume percentage of mobile phase B linearly increases to 20%; from 5 minute to 18 minute, the volume percentage of mobile phase A linearly decreases to 70%, and the volume percentage of mobile phase B linearly increases to 30%; from 18 minute to 40 minute, the volume percentage of mobile phase A is 70%, and the volume percentage of mobile phase B is 30 %; from 40 minutes to 48 minutes, the volume percentage of mobile phase A linearly decreases to 60%, and the volume percentage of mobile phase B linearly increases to 40%; from 48 minutes to 52 minutes, the volume percentage of mobile phase A linearly increases to 80%, and the volume percentage of mobile phase B linearly decreases to 20%; from 52 minutes to 55 minutes, the volume percentage of mobile phase A linearly increases to 90%, and the volume percentage of mobile phase B linearly decreases to 10%; from 55 minutes to 65 minutes, the volume percentage of mobile phase A is 90%, and the volume percentage of mobile phase B is 10%.

7. The method according to claim 1, characterized in that Step 3) The chromatographic column uses octadecylsilane bonded silica gel as a filler or a chromatographic column with equivalent performance. The chromatographic column model is ZORBAX SB-C18, with a specification of 4.6 mm×250 mm, 5 μm.

8. The method according to claim 1, characterized in that Step 4) Detection was performed using a wavelength of 238 nm and the column temperature of the chromatographic column was 30-40°C.

9. The method according to claim 8, characterized in that The column temperature was 35°C.

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