CN115980217A - Furosemide and control method for determining content of degradation impurities in injection of furosemide - Google Patents
Furosemide and control method for determining content of degradation impurities in injection of furosemide Download PDFInfo
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- CN115980217A CN115980217A CN202211673331.2A CN202211673331A CN115980217A CN 115980217 A CN115980217 A CN 115980217A CN 202211673331 A CN202211673331 A CN 202211673331A CN 115980217 A CN115980217 A CN 115980217A
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Abstract
The invention discloses a furosemide and an analysis method for determining the content of degradation impurities in injection thereof. The method comprises dissolving furosemide in tetrahydrofuran, and measuring by high performance liquid chromatography with evaporative light scattering detector. And with C 18 And the chromatographic column takes a mixed solution of a buffer salt solution and tetrahydrofuran as a mobile phase. The buffered salt solution is an ammonium acetate solution. The invention provides a method for rapidly determining degraded impurities in furosemide,the method has the advantages of strong specificity, high sensitivity, good accuracy and simple and convenient operation, and can effectively control the quality of the furosemide.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting the content of degraded impurities in furosemide and injection thereof by using a high performance liquid chromatography, which is used for detecting the content of photodegradable impurities M (2- [ (furan-2-ylmethyl) -amino ] -4-hydroxy-3-methylene amino-5-sulfamoylmethyl benzoate) in furosemide raw materials and injection thereof.
Background
Furosemide, british name Furosemide. Is widely used for treating edema related to heart failure in clinicCan be used alone or in combination with other antihypertensive drugs for treating hypertension. Molecular formula C 12 H 11 ClN 2 O 5 S, molecular weight 330.75. The structural formula is shown in figure 1.
Furosemide and preparations thereof are mostly stored in a dark place. It was also found during the stability study that furosemide is susceptible to degradation under light conditions. Furosemide produces a variety of degradation impurities under light conditions. Mainly wherein the degradation impurity M is one of the main photodegradation impurities, and is easy to generate in an acidic solution. The molecular formula of degrading the impurity M is C 14 H 15 N 3 O 6 S, the molecular weight is 353.07, and the structural formula is shown in figure 2.
According to the requirements of the State food and drug administration, related impurities are controlled. According to the technical requirements for evaluating the consistency of the quality and the curative effect of the imitation drug of the chemical injection, the degradation products of the preparation are mainly researched, wherein the degradation products comprise the degradation products of the raw material medicines or the reaction products of the raw material medicines and auxiliary materials and/or inner packaging materials. Tetrahydrofuran has large ultraviolet absorption, and easily influences a base line under ultraviolet detection, so that a chromatogram with a poor base line is formed, and sample detection is influenced. Therefore, the product adopts a novel evaporative light scattering detector which can well support gradient elution and can eliminate the influence of the change of the flow matching ratio on the baseline. Therefore, an evaporative light scattering detector is selected.
The degradation impurity M has low degradation degree in the furosemide raw material, is easier to degrade in the injection and is the limit for strictly controlling the degradation impurity in the raw material. The injection concentration of furosemide needs to be increased.
Furosemide is insoluble in water, slightly soluble in ethanol and readily soluble in tetrahydrofuran. In order to increase the solubility of furosemide, the interference of solvent effect on the detection is reduced. Tetrahydrofuran was chosen as solvent for the process.
The method in the research adopts the liquid chromatography, uses the evaporative light scattering detector for control, has simple operation, has the sensitivity of 20ppm and meets the minimum requirement.
Disclosure of Invention
The invention aims to provide a method for separating and measuring photodegradable impurities M in furosemide and injection thereof by liquid chromatography, which adopts an evaporative light scattering detector to measure and adopts tetrahydrofuran as a solvent. The method can effectively achieve quantitative detection of the degradation impurity M through verification. Thereby realizing the effective control of the quality of the furosemide raw material and the preparation thereof. The method has high sensitivity and specificity.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for controlling the content determination of degraded impurities in furosemide and injection thereof is characterized in that: detecting by high performance liquid chromatography with an evaporative light scattering detector under the following detection conditions:
a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler, wherein the specification is 4.6 multiplied by 250mm and 5 mu m;
mobile phase: a buffered salt solution and tetrahydrofuran;
a detector: an evaporative light scattering detector.
The buffer salt solution is one of an ammonium formate solution and an ammonium acetate solution.
The concentration of the buffer salt solution is 0.1-0.2 mol/L.
The flow rate of the mobile phase is 0.8-1.2 ml/min.
The temperature of the chromatographic column is 30-40 ℃.
The drift tube temperature of the detector is 80-90 ℃.
The gas flow rate of the detector is 1.5-2.0L/min.
The specific method for measuring the content of the degraded impurity M in the furosemide is as follows:
1. instruments and reagents
Agilent 1100 hplc, tetrahydrofuran (chromatographic grade), ammonium acetate (analytical grade), ammonium formate (analytical grade), SATORIOUS analytical balance.
Diluent agent: tetrahydrofuran.
Buffer salt solution: ammonium formate or ammonium acetate was dissolved in 1000ml of water.
Control solution: taking a proper amount of impurity M reference substance, quantitatively diluting with tetrahydrofuran, and shaking up to obtain reference substance solution.
2. And (3) determination of a sample:
(1) Preparation of raw material samples: and (4) operating in dark. And taking a proper amount of the furosemide sample, and precisely weighing. Adding tetrahydrofuran for dissolving, quantitatively diluting with tetrahydrofuran to scale, and shaking.
(2) Preparation of preparation samples: taking a proper amount of furosemide injection (about 50mg of furosemide), diluting with tetrahydrofuran by 10 times, and shaking up to obtain the furosemide injection.
(3) Chromatographic conditions are as follows:
mobile phase: eluting with water phase buffer salt solution and organic phase tetrahydrofuran.
High performance liquid chromatography is adopted for determination, and an evaporative light scattering detector is adopted.
(4) Specificity test
Under the chromatographic condition, respectively taking a blank solvent, an impurity M reference substance solution, a furosemide test substance solution, an injection test substance solution and other known impurity solutions in sequence, and respectively carrying out sample injection detection, wherein the separation degree of the impurity M and an adjacent chromatographic peak is more than 1.5. The blank solvent, the known impurities and the furosemide peak do not interfere with the detection of the impurity M, and the specificity meets the requirement.
(5) Linearity and range
Preparing impurity M solutions with different concentrations, wherein the range is a solution with the quantitative limit of 200% of the limit concentration. The peak area of the impurity M was recorded. And drawing a linear regression line by taking the concentration as an abscissa and taking the peak area as an ordinate. Obtaining a linear regression equation: y =64765x-138.95, R 2 =1.0000. The y-axis intercept is 0.1%. Meets the requirements.
(6) Quantitative limit and detection limit
Under the chromatographic conditions, the limit of quantitation is determined according to the response concentration when the signal-to-noise ratio of chromatographic peaks is 10, the detection limit is determined according to the response concentration when the signal-to-noise ratio is 3.
(7) Precision test
Preparing one part of impurity M reference substance solution with quantitative limit concentration, continuously injecting 6 needles according to the chromatographic conditions, recording peak area, and calculating retention time and RSD% value of the peak area. The retention time RSD% value was 0.5%, less than 2%, and the peak area RSD% value was 1.8%, less than 5%. Meets the requirements.
(8) Accuracy of
Weighing 13 parts of sample, wherein 1 part of sample is the solution of the test sample without the added standard, and preparing the solution of the test sample normally according to the preparation method of the solution of the test sample. The other 12 parts were divided into 4 groups of 3 parts each. The standard addition is carried out according to the limit concentration of the impurity M, the limit concentration of 80%, 100% and 120%, and 3 parts of standard addition samples of each concentration are prepared in parallel. All samples were measured according to the chromatographic conditions described above. The results are as follows:
note: N/A indicates not applicable.
(9) Stability of solution
And respectively placing the reference substance solution and the test substance solution of the impurity M at room temperature in a dark condition for 48 hours. The peak area of the impurity M was measured, and the SD% value of the peak area at each time and the peak area at 0 time was calculated. The result shows that the SD% value of the peak area of the impurity M control solution is 1.6% and less than 2% within 48 hours under the conditions of room temperature and light shielding. Indicating that the control solution of impurity M is in a steady state within 48 hours. The impurity M control solution can be used at room temperature for 48 hours in the dark. The test solution was left at room temperature in a dark state for 48 hours, and no impurity M was detected. Therefore, the test solution is stable within 48 hours at room temperature in a dark state.
The method has the advantages of good specificity, high sensitivity, wide linear range, good solution stability and good system applicability of the instrument. Provides an effective method for practically determining photodegradable impurities M in the furosemide raw material and the preparation thereof.
The invention has the following positive effects: the method adopts the high performance liquid chromatography and uses the evaporative light scattering detector to measure the furosemide photodegradation impurity M, thereby realizing the content measurement of the furosemide and the impurity M in the injection thereof and finally ensuring the safety and the effectiveness of the furosemide and the preparation thereof.
1. Selection of mobile phase buffer salt species and salt concentration
Ammonium formate, ammonium acetate, ammonium nitrate and ammonium dihydrogen phosphate are taken into consideration on the basis of an evaporative light scattering detector and the pKa value of a detected compound. The same photo-destructive sample solution was tested using a salt solution of the same molar concentration. Experiments show that ammonium formate and ammonium acetate can be used as mobile phase buffer salts of the furosemide photodegradation impurities M. On the basis of the two salts, by inspecting the detection conditions of different concentrations on the furosemide photodegradation impurities, a buffer salt solution of 0.1-0.2 mol/L ammonium formate or ammonium acetate solution is selected as a mobile phase, and the specific test detection conditions are as follows:
the salt concentration selection test cases were as follows:
2. column temperature, mobile phase flow rate and selection of chromatographic column
The inventors examined the detection of this photodegradation impurity M at different column temperatures and flow rates of the mobile phase. The method comprises the following specific steps:
| column temperature of chromatography column, deg.C | Photodegradation of impurity |
| 20 | Not detected out |
| 30 | 0.521 |
| 40 | 0.520 |
| 50 | Has interference and is not detected |
| Flow rate, mL/min | Photodegradation of impurity detection conditions% |
| 0.5 | Not detected out |
| 0.8 | 0.521 |
| 1.0 | 0.522 |
| 1.2 | 0.521 |
| 1.5 | Has interference and is not detected |
According to results, the method for analyzing the content of the degraded impurities in the furosemide and the injection thereof is characterized in that the column temperature of the chromatographic column is 30-40 ℃, and the flow velocity of the mobile phase is 0.8-1.2 ml/min.
3. Selection of drift tube temperature and gas flow rate
Specifically, the inventor screens the drift tube temperature and the gas flow rate according to the characteristics of the evaporative light scattering detector, and specifically includes the following steps:
| drift tube temperature,. Deg.C | Photodegradation of impurity detection conditions% | Gas flow rate, L/min | Photodegradation of impurity detection conditions% |
| 70 | Not detected out | 1.0 | Not detected out |
| 80 | 0.521 | 1.5 | 0.522 |
| 90 | 0.522 | 2.0 | 0.521 |
| 100 | Not detected out | 3.0 | Not detected out |
The results show that the detection conditions of the evaporative light scattering detector include a drift tube temperature of 80 to 90 ℃ and a gas flow rate of 1.5 to 2.0L/min.
Drawings
FIG. 1: furosemide structure formula.
FIG. 2 is a drawing: the structural formula of the impurity M is shown in the figure.
FIG. 3 is a drawing: the impurity M control solution chromatogram obtained in example 1 according to the invention.
FIG. 4 is a drawing: the furosemide test sample solution chromatogram obtained in the embodiment 1 of the invention is shown.
Detailed Description
The following examples are intended to illustrate the invention, but not to limit the scope of the invention.
Example 1
An instrument high performance liquid chromatograph: agilent, 1100, evaporative light scattering detector.
And (3) chromatographic column: thermo HYPERSIL BDS C 18 4.6×250mm，5μm。
Mobile phase: using buffered salt solution as aqueous phase (weighing 7.7g ammonium acetate in 1000ml water); tetrahydrofuran is used as an organic phase.
Elution gradient:
| time (minutes) | Aqueous phase (%) | Organic phase (%) |
| 0 | 90 | 10 |
| 16 | 90 | 10 |
| 18 | 20 | 80 |
| 32 | 20 | 80 |
| 33 | 90 | 10 |
| 45 | 90 | 10 |
Flow rate: 1.0ml/min.
Column temperature: at 30 ℃.
Drift tube temperature: at 90 ℃.
Gas flow rate: 2.0L/min.
Sample introduction volume: 20 μ l.
Diluent (b): tetrahydrofuran.
Preparing a sample solution: taking a proper amount of impurity M reference substance solution, dissolving with a diluent, and quantitatively diluting to obtain a solution of about 10 μ g per 1ml as reference substance solution; taking 50mg of the product, precisely weighing, placing in a 20ml measuring flask, adding a diluent to dissolve, and then fixing the volume to a scale to obtain a test solution.
Precisely measuring reference solution and sample solution, respectively injecting into liquid chromatograph, and recording chromatogram as shown in figure 1 and figure 2. The impurity M is not detected according to the calculation of an external standard method.
Example 2.
The instrument comprises the following steps: the same as in example 1.
A chromatographic column: HILIC C 18 4.6×250mm，5μm。
Flow rate: 1.2ml/min.
Column temperature: at 40 ℃.
Temperature of the drift tube: 85 ℃.
Gas flow rate: 1.8L/min.
Elution gradient, sample volume and diluent were as in example 1.
Mobile phase: using a buffered salt solution as an aqueous phase (11 g of ammonium acetate was weighed into 1000ml of water); tetrahydrofuran is used as an organic phase.
Preparing a sample solution: taking a proper amount of impurity M reference substance solution, dissolving by using a diluent, and quantitatively diluting to prepare a solution of which each 1ml is about 20 mu g as the reference substance solution; taking 5ml of furosemide injection, placing the furosemide injection into a 10ml measuring flask, quantitatively diluting the diluent to a scale mark, and shaking up to obtain a test solution.
Precisely measuring the reference solution and the sample solution, respectively injecting into a liquid chromatograph, recording chromatogram, and measuring the content of impurity M to be 0.005% according to an external standard method.
Claims (5)
1. An analysis method for measuring the content of degradation impurities in furosemide and injection thereof is characterized in that the content of the photodegradation impurities M (2- [ (furan-2-ylmethyl) -amino ] -4-hydroxy-3-methylene amino-5-sulfamoylmethyl benzoate) in furosemide and injection thereof is measured by using a high performance liquid chromatography and an evaporation light scattering detector.
2. The assay of claim 1 wherein the furosemide sample is diluted with tetrahydrofuran.
3. The analytical method according to claim 1, wherein the content of the degraded impurities is determined by high performance liquid chromatography using an evaporative light scattering detector.
4. The analytical method according to claim 1, wherein octadecylsilane bonded silica (C) is used 18 ) Is a filler chromatographic column.
5. The analysis method according to claim 1, characterized in that it comprises the following main contents: the mobile phase water phase is ammonium formate or ammonium acetate buffer solution, and the salt concentration is 0.1 to 0.2mol/L; the column temperature of the chromatographic column is 30 to 40 ℃; the flow rate of the mobile phase is 0.8 to 1.2ml/min; the temperature of a drift tube of the detector is 80 to 90 ℃; the gas flow rate of the detector is 1.5 to 2.0L/min.
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