Degradation impurity of furosemide and preparation method thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a furosemide degradation impurity and a preparation method thereof. The preparation method comprises the following steps: dissolving furosemide in purified water, adding a regulator, and adding a sodium hydroxide aqueous solution to regulate the pH value to obtain a mixed solution; carrying out illumination degradation on the mixed solution, and then carrying out rough filtration and fine filtration to obtain a sample solution; separating and purifying the sample solution through reversed-phase liquid chromatography, and collecting a target impurity peak solution; and carrying out reduced pressure distillation on the target impurity peak solution, and freeze-drying to obtain the furosemide degradation impurity. The purity of the prepared furosemide degradation impurity reaches 97% or above, and the furosemide degradation impurity can be used as a reference substance for quality research and quality control of furosemide.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a furosemide degradation impurity and a preparation method thereof. Background Art

[0002] Furosemide belongs to sulfonamide diuretics and entered clinical use in 1964. Its main action site is the NKCC2 membrane protein in the thick ascending limb of the renal tubule loop of Henle. It is a compound that is insoluble in water but is stable enough in gastric juice, duodenal juice, bile, and urine. Furosemide can directly increase the excretion of sodium, potassium, and chloride, thereby increasing the exchange of water and ions in the renal tubules, which is beneficial to excreting excess substances and metabolites in the body through urine. In addition, furosemide also has a vasodilating effect, which is related to the inhibition of the activity of prostaglandin-degrading enzymes and the increase in the secretion of prostaglandin E2. Therefore, in addition to diseases with increased blood volume such as heart failure, pulmonary edema, and hypertension, as well as metabolic abnormalities such as hyperkalemia, hypercalcemia, dilutional hyponatremia, syndrome of inappropriate antidiuretic hormone secretion, and acute drug poisoning, it can also play a role in dilating renal blood vessels and reducing renal vascular resistance. This mechanism can be used to prevent acute renal failure and acute kidney injury, increase renal blood flow, and slow down the degree of decline in renal function. Nebulized furosemide can treat dyspnea and also has the effect of treating bronchial asthma.

[0003] In the modern pharmaceutical industry, to ensure drug safety, controlling the amount of impurities in drugs is an important item in the field of drug quality control. During the drug R & D and consistency evaluation processes, it is necessary to analyze, classify, and study each possible impurity one by one and take appropriate measures for control.

[0004] During the R & D, production, and storage of furosemide, various impurities will also be generated. Shinji Katsura et al. in Identification of Furosemide Photodegradation Products in Water–Acetonitrile Mixture, Chem. Pharm. Bull. 63(8), 617–627 (2015) studied the photodegradation products of furosemide in a water-acetonitrile mixed solution, mainly involving four products, and the reaction equations are as follows: 。

[0005] Studies on the Photolysis and Hydrolysis of Furosemide in Aqueous Solution, by Naomi Yagi et al., Chem. Pharm. Bull. 39(2), 454~457 (1991) studied the photolysis and hydrolysis of furosemide in aqueous solution, mainly involving Degradation Product 1 and Degradation Product 3 in the above reaction equations.

[0006] Chinese Patent CN116854651A discloses a preparation method of furosemide photo-degradation impurities. The specific preparation steps are as follows: Step S1, take furosemide and place it in a reaction vessel, and dilute it with glacial acetic acid and acetonitrile aqueous solution as diluents to 0.5~5 mg / ml; Step S2, shake the mixed solution obtained in Step S1 and place it in a light box with a light intensity of 6000~8000 Lx, irradiate for 21 - 24 h to obtain a degradation solution; Step S3, subject the degradation solution obtained in Step S2 to preparative liquid chromatography, and collect the eluate with a retention time of 6.97 ± 0.5 min at a wavelength of 272 nm, and that is what is obtained. The furosemide photo-degradation impurities prepared in this patent involve Degradation Product 3 in the above reaction equations.

[0007] In the research of furosemide preparations in our company, a new unknown impurity was discovered. This impurity even became the largest impurity in the preparation under certain conditions. Through analysis and experiments, it was judged that this impurity should be a photo-degradation impurity, but this impurity is different from the impurities mentioned in the above literature and has not been reported in the literature.

[0008] To ensure the safety of drug use, successfully develop a new furosemide preparation process, and ensure the quality of furosemide preparations, it is necessary to clarify the structure of this unknown impurity, identify the source and category of the impurity, so as to provide a basis for determining appropriate preparation processes and storage conditions in the research of furosemide preparations. Therefore, we studied the preparation method of this impurity, prepared a target impurity in a researchable amount, and carried out structure confirmation based on this. Summary of the Invention

[0009] The purpose of the present invention is to provide a furosemide degradation impurity with a purity of more than 97%, which can be used as a reference substance for quality control; the present invention also provides a preparation method of the furosemide degradation impurity.

[0010] The furosemide degradation impurity described in the present invention has the following structural formula: .

[0011] The preparation method of the furosemide degradation impurity described in the present invention includes the following steps: (1) Dissolve furosemide in purified water, add a regulator, and then add an aqueous sodium hydroxide solution to adjust the pH to obtain a mixed solution; (2) After subjecting the mixed solution to photocatalytic degradation, it is subjected to coarse filtration and then fine filtration to obtain a sample solution; (3) The sample solution is separated and purified by reverse-phase liquid chromatography, and the target impurity peak solution is collected; (4) The target impurity peak solution is subjected to vacuum distillation and freeze-drying to obtain furosemide degradation impurities.

[0012] In step (1), the regulator is sodium chloride or potassium chloride.

[0013] In step (1), the mass concentration of the sodium hydroxide aqueous solution is 10-20%.

[0014] In step (1), the ratio of furosemide, purified water and the regulator is 1:50-100:0.5-1.0, where furosemide and the regulator are in g, and purified water is in ml.

[0015] In step (1), the pH is adjusted to 6.5-7.5.

[0016] In step (2), the light intensity is 4000-5000 Lux, and the light exposure time is 48-72 hours; the filter membrane used for coarse filtration is a neutral filter paper, and the filter membrane used for fine filtration is a water-based filter membrane, preferably a 0.45 µm water-based filter membrane.

[0017] In step (3), the chromatographic conditions for reverse-phase liquid chromatography are as follows: Chromatographic column: Kromasil 100-5-C18, Φ30×250 mm Detector: Ultraviolet-visible light detector Detection wavelength: 272 nm Mobile phase and gradient: 0 min, 50% methanol - 50% aqueous acetic acid solution; 8 min, 50% methanol - 50% aqueous acetic acid solution; 9 min, 90% methanol - 10% aqueous acetic acid solution; 14 min, 90% methanol - 10% aqueous acetic acid solution; 14.5 min, 50% methanol - 50% aqueous acetic acid solution; 21 min, 50% methanol - 50% aqueous acetic acid solution; where the volume concentration of the aqueous acetic acid solution is 1‰ Flow rate: 30 ml / min Injection volume: 4.5-5.5 ml.

[0018] In step (3), the retention time of the target impurity peak is 17.0-17.7 min.

[0019] In step (4), the temperature for vacuum distillation is 25-35 °C.

[0020] The present invention uses furosemide as the starting material, dissolves it in water, adds a regulator to adjust the ion concentration, adjusts the pH, and then produces the target impurity through light irradiation. Subsequently, the furosemide degradation impurity is obtained through reverse-phase liquid chromatography separation and purification.

[0021] The reaction principle of the present invention is as follows: 。

[0022] The beneficial effects of the present invention are as follows: 1. The furosemide degradation impurity prepared by the present invention is a photo-degradation impurity of furosemide. Therefore, in order to control the quality of furosemide, light irradiation should be avoided during the production and storage of furosemide to reduce the generation of light-degraded impurities.

[0023] 2. The purity of the furosemide degradation impurity prepared by the present invention reaches over 97%, and it can be used as a reference substance for the quality research and quality control of furosemide. The present invention has positive significance for the formulation, process development, quality research, and storage condition research of the preparation.

[0024] 3. The raw materials used in the present invention are easily available, and the process parameters are controllable. Description of the Drawings

[0025] Figure 1 is the HPLC chart of the sample solution in Example 1.

[0026] Figure 2 is the HPLC chart of the furosemide degradation impurity in Example 1.

[0027] Figure 3 is the mass spectrometry chart of the furosemide degradation impurity in Example 1.

[0028] Figure 4 is the NMR-H spectrum chart of the furosemide degradation impurity in Example 1.

[0029] Figure 5 is the NMR-C spectrum chart of the furosemide degradation impurity in Example 1.

[0030] Figure 6 is the COSY spectrum chart of the furosemide degradation impurity in Example 1.

[0031] Figure 7 is the HMBC spectrum chart of the furosemide degradation impurity in Example 1.

[0032] Figure 8 is the HSQC spectrum chart of the furosemide degradation impurity in Example 1.

[0033] Figure 9 is the DEPT135 spectrum chart of the furosemide degradation impurity in Example 1. Detailed Embodiments

[0034] The present invention will be further described below in conjunction with embodiments.

[0035] Example 1 (1) Weigh 0.75 g of furosemide raw material in a reaction flask, add 50 ml of purified water and 0.56 g of sodium chloride, stir to dissolve, and adjust the pH to 7.0 with 15 wt.% sodium hydroxide aqueous solution to obtain a mixed solution; (2) Put the mixed solution into a transparent borosilicate glass bottle, seal the bottle mouth, place it in a light box for 60 hours under 4500 Lux light. First, filter the light-exposed solution roughly with a neutral filter paper, and then filter it precisely with a 0.45 µm water-based filter membrane to remove insoluble substances to obtain a sample solution. The HPLC chromatogram of the sample solution is shown in Figure 1 , and the HPLC content of the target impurity is 35.34%; (3) Separate and purify the sample solution by reverse-phase liquid chromatography, and collect the solution of the target impurity peak; The chromatographic conditions of the reverse-phase liquid chromatography are as follows: Chromatographic column: Kromasil 100-5-C18, Φ30×250 mm Detector: Ultraviolet-visible light detector Detection wavelength: 272 nm The mobile phase and gradient are shown in Table 1:

[0036] Flow rate: 30 ml / min Injection volume: 5 ml (4) Evaporate methanol from the solution of the target impurity peak under reduced pressure at 30 °C, and then freeze-dry to obtain furosemide degradation impurity, with an HPLC content of 98.49%; ESI-MS: m / z = 267.05 ([M-H] - ); 1 1H-NMR(DMSO-d6, 298 K): δ ppm : 4.23 (d, J= 5.9 Hz, 2H), 6.16 (m, overlap , 1H), 6.28 (d, J= 3.1 Hz, 1H), 6.39 (dd, J= 3.1, 1.9Hz, 1H), 6.58 (br, 2H), 6.73 (t, J= 5.9 Hz, 1H), 7.31 (d, J= 9.2 Hz, 1H), 7.57 (m,1H), 10.05 (br, 1H); 13 13C-NMR(DMSO-d6, 298 K): δ ppm: 39.8, 99.0, 103.5, 107.5, 110.9, 117.3, 129.1, 142.6, 153.0, 153.2, 156.2.

[0037] The HPLC chromatogram of the furosemide degradation impurity is shown in Figure 2 , and the mass spectrum is shown in Figure 3 , the NMR-H spectrum is shown in Figure 4 , the NMR-C spectrum is shown in Figure 5 , the COSY spectrum is shown in Figure 6 , the HMBC spectrum is shown in Figure 7 , the HSQC spectrum is shown in Figure 8 , the DEPT135 spectrum is shown in Figure 9 .

[0038] Example 2 (1) Weigh 0.50 g of furosemide raw material drug into a reaction flask, add 50 ml of purified water and 0.50 g of sodium chloride, stir to dissolve, and adjust the pH to 7.0 with 10 wt.% sodium hydroxide aqueous solution to obtain a mixed solution; (2) Put the mixed solution into a transparent borosilicate glass bottle, seal the bottle mouth, place it in a light box at 4000 Lux for 72 hours. First, filter the light-exposed solution roughly with a neutral filter paper, and then filter it precisely with a 0.45 µm aqueous filter membrane to remove insoluble substances to obtain a sample solution, and the HPLC content of the target impurity is 37.25%; (3) Separate and purify the sample solution by reverse-phase liquid chromatography, and collect the target impurity peak solution; Injection volume: 4.5 ml, and other chromatographic conditions of reverse-phase liquid chromatography are the same as those in Example 1; (4) Evaporate methanol from the target impurity peak solution under reduced pressure at 25 °C, and then freeze-dry to obtain furosemide degradation impurity, and the HPLC content is 98.67%.

[0039] Example 3 (1) Weigh 1.00 g of furosemide raw material drug into a reaction flask, add 50 ml of purified water and 0.50 g of sodium chloride, stir to dissolve, and adjust the pH to 6.5 with 20 wt.% sodium hydroxide aqueous solution to obtain a mixed solution; (2) Put the mixed solution into a transparent borosilicate glass bottle, seal the bottle mouth, place it in a light box at 5000 Lux for 48 hours. First, filter the light-exposed solution roughly with a neutral filter paper, and then filter it precisely with a 0.45 µm aqueous filter membrane to remove insoluble substances to obtain a sample solution, and the HPLC content of the target impurity is 33.48%; (3) Separate and purify the sample solution by reverse-phase liquid chromatography, and collect the target impurity peak solution; Injection volume: 5.5 ml, and other chromatographic conditions of reverse-phase liquid chromatography are the same as those in Example 1; (4)The target impurity peak solution was evaporated to remove methanol under reduced pressure at 35°C, and then lyophilized to obtain furosemide degradation impurities with an HPLC content of 97.28%.

[0040] Example 4 (1)Weigh 0.75 g of furosemide raw material drug into a reaction flask, add 50 ml of purified water and 0.56 g of potassium chloride, stir to dissolve, and adjust the pH to 7.0 with 10 wt.% sodium hydroxide aqueous solution to obtain a mixed solution. (2)The mixed solution was filled into a transparent borosilicate glass bottle, the bottle mouth was sealed, and it was placed in a light box for illumination at 4800 Lux for 72 hours. The illuminated solution was first filtered roughly with a neutral filter paper, and then filtered precisely with a 0.45 µm water-based filter membrane to remove insoluble substances, obtaining a sample solution with a target impurity HPLC content of 36.28%. (3)The sample solution was separated and purified by reverse-phase liquid chromatography, and the target impurity peak solution was collected. The chromatographic conditions of the reverse-phase liquid chromatography were the same as those in Example 1. (4)The target impurity peak solution was evaporated to remove methanol under reduced pressure at 35°C, and then lyophilized to obtain furosemide degradation impurities with an HPLC content of 98.21%.

[0041] Example 5 (1)Weigh 0.50 g of furosemide raw material drug into a reaction flask, add 50 ml of purified water and 0.50 g of sodium chloride, stir to dissolve, and adjust the pH to 7.0 with 15 wt.% sodium hydroxide aqueous solution to obtain a mixed solution. (2)The mixed solution was filled into a transparent borosilicate glass bottle, the bottle mouth was sealed, and it was placed in a light box for illumination at 4500 ± 500 Lux for 60 hours. The illuminated solution was first filtered roughly with a neutral filter paper, and then filtered precisely with a 0.45 µm water-based filter membrane to remove insoluble substances, obtaining a sample solution with a target impurity HPLC content of 35.69%. (3)The sample solution was separated and purified by reverse-phase liquid chromatography, and the target impurity peak solution was collected. The chromatographic conditions of the reverse-phase liquid chromatography were the same as those in Example 1. (4)The target impurity peak solution was evaporated to remove methanol under reduced pressure at 30°C, and then lyophilized to obtain furosemide degradation impurities with an HPLC content of 98.32%.

[0042] Example 6 (1)Weigh 0.75 g of furosemide raw material drug into a reaction flask, add 50 ml of purified water and 0.40 g of sodium chloride, stir to dissolve, and adjust the pH to 7.5 with 10 wt.% sodium hydroxide aqueous solution to obtain a mixed solution. (2) The mixed solution was filled into a transparent borosilicate glass bottle, the bottle mouth was sealed, and it was placed in a light box for 60 hours under 4600 Lux light. The solution after light exposure was first filtered roughly with a neutral filter paper, and then filtered precisely with a 0.45 µm aqueous filter membrane to remove insoluble substances, obtaining a sample solution with the HPLC content of the target impurity being 34.25%; (3) The sample solution was separated and purified by reverse-phase liquid chromatography, and the solution of the target impurity peak was collected; The chromatographic conditions of the reverse-phase liquid chromatography were the same as those in Example 1; (4) The solution of the target impurity peak was evaporated to remove methanol under reduced pressure at 30 °C, and then freeze-dried to obtain furosemide degradation impurity with the HPLC content being 98.25%.

[0043] Content detection experiment of furosemide degradation impurity in furosemide injection: Using the furosemide degradation impurity prepared in Example 1 as a reference substance, the content of furosemide degradation impurity in furosemide injection was detected.

[0044] Detection method: It was determined according to the high performance liquid chromatography method (General Principles 0512, Volume IV, Chinese Pharmacopoeia 2020 Edition), and the operation was carried out under light protection.

[0045] Mixed solvent: Take 22 ml of glacial acetic acid, add acetonitrile-water (1:1) to 1000 ml, and mix well.

[0046] Test solution: Accurately measure an appropriate amount of furosemide injection, and quantitatively dilute it with the mixed solvent to prepare a solution containing 1 mg of furosemide per 1 ml.

[0047] Impurity reference substance solution: Accurately weigh an appropriate amount of furosemide degradation impurity prepared in Example 1, dissolve it with the mixed solvent and quantitatively dilute it to prepare a solution containing 2 µg of furosemide degradation impurity per 1 ml as the impurity reference substance solution.

[0048] Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler (Agilent ZORBAX Eclipse XDB-C18, 4.6 mm × 250 mm, 5 µm or a chromatographic column with equivalent efficiency); use water (adjust the pH value to 3.0 with phosphoric acid) as mobile phase A, and use tetrahydrofuran as mobile phase B for gradient elution. The gradient elution program is shown in Table 2; the column temperature is 40 °C; the flow rate is 1.0 ml per minute; the detection wavelength is 272 nm; the injection volume is 20 μl.

[0049]

[0050] Determination method: Accurately measure the test solution and the impurity reference substance solution, and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0051] Test results: The content of furosemide degradation impurities was calculated by the external standard method. Six test solution samples were detected in parallel, and the content of furosemide degradation impurities was between 0.02% and 0.05%.

[0052] The method for preparing furosemide degradation impurities provided by the present invention is simple, and can be used as a reference substance to effectively detect furosemide degradation impurities in furosemide drugs, further strengthening the quality control of furosemide drugs.

[0053] Toxicity test of impurities: The LD50 of rats by oral administration of the furosemide degradation impurities prepared in Example 1 was determined, and the LD50 of rats by oral administration of furosemide degradation impurities was 4550 mg / kg; the LD50 of rats by intravenous injection of the furosemide degradation impurities prepared in Example 1 was determined, and the LD50 of rats by intravenous injection of furosemide degradation impurities was 2650 mg / kg.

[0054] The present invention has been described in detail above in conjunction with the embodiments, but the content described is only the preferred embodiment of the present invention and cannot be considered as limiting the scope of implementation of the present invention. Any equivalent changes and improvements made according to the scope of the application of the present invention shall still fall within the scope covered by the patent of the present invention.

Claims

1. A furosemide degradation impurity, characterized in that The structural formula is as follows: 。 2. The preparation method of the furosemide degradation impurity according to claim 1, characterized in that It includes the following steps: (1) Dissolve furosemide in purified water, add a regulator, and then add an aqueous sodium hydroxide solution to adjust the pH to obtain a mixed solution; (2) After subjecting the mixed solution to light degradation, perform coarse filtration and fine filtration to obtain a sample solution; (3) Separate and purify the sample solution by reverse-phase liquid chromatography, and collect the target impurity peak solution; (4) Subject the target impurity peak solution to vacuum distillation and lyophilization to obtain furosemide degradation impurities.

3. The preparation method of the furosemide degradation impurity according to claim 2, characterized in that In step (1), the regulator is sodium chloride or potassium chloride.

4. The preparation method of the furosemide degradation impurity according to claim 2, wherein In step (1), the mass concentration of the aqueous sodium hydroxide solution is 10-20%.

5. The preparation method of the furosemide degradation impurity according to claim 2, wherein In step (1), the ratio of furosemide, purified water, and the regulator is 1:50-100:0.5-1.0, where furosemide and the regulator are in g, and purified water is in ml.

6. The preparation method of the furosemide degradation impurity according to claim 2, wherein In step (1), adjust the pH to 6.5-7.

5.

7. The preparation method of the furosemide degradation impurity according to claim 2, characterized in that In step (2), the light intensity is 4000-5000 Lux, and the light exposure time is 48-72 hours; the filter membrane used for coarse filtration is a neutral filter paper, and the filter membrane used for fine filtration is a water-based filter membrane.

8. The preparation method of the furosemide degradation impurity according to claim 2, wherein In step (3), the chromatographic conditions for reverse-phase liquid chromatography are as follows: Chromatographic column: Kromasil 100-5-C18, Φ30×250mm Detector: Ultraviolet-visible light detector Detection wavelength: 272nm Mobile phase and gradient: 0 min, 50% methanol - 50% aqueous acetic acid solution; 8 min, 50% methanol - 50% aqueous acetic acid solution; 9 min, 90% methanol - 10% aqueous acetic acid solution; 14 min, 90% methanol - 10% aqueous acetic acid solution; 14.5 min, 50% methanol - 50% aqueous acetic acid solution; 21 min, 50% methanol - 50% aqueous acetic acid solution; where the volume concentration of the aqueous acetic acid solution is 1‰ Flow rate: 30 ml / min Injection volume: 4.5-5.5 ml.

9. The preparation method of the furosemide degradation impurity according to claim 2, characterized in that In step (3), the retention time of the target impurity peak is 17.0-17.7 min.

10. The preparation method of the furosemide degradation impurity according to claim 2, characterized in that In step (4), the temperature for vacuum distillation is 25-35°C.

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