Method for detecting mutagenic impurities in furosemide injection through high performance liquid chromatography

Abstract

The invention discloses a method for detecting the content of mutagenic impurities in furosemide injection, belongs to the technical field of pharmaceutical analysis, and relates to a method for detecting mutagenic impurities in furosemide injection through high performance liquid chromatography, which comprises the following steps: (1) setting high performance liquid chromatography conditions; (2) sample preparation: a solvent: acetonitrile-water = 20: 80; a blank auxiliary material solution; a test solution; a reference solution; and (3) respectively injecting into a liquid chromatograph, recording chromatograms, and calculating by peak area according to an external standard method. According to the method disclosed by the invention, two mutagenic impurities can be simultaneously detected in one detection method, and the method is strong in specificity, high in sensitivity, good in linear relation, high in accuracy and good in detection durability and stability; the method can accurately reflect the real content of two mutagenic impurities furfuryl alcohol and furfural in the furosemide injection, effectively reduces the detection cost, effectively improves the quality control of the furosemide injection, ensures the quality and quantity safety of drugs, and reduces the clinical toxic and side effects possibly brought by the drugs.

Description

Technical Field

[0001] The present invention relates to a high performance liquid chromatography detection method, and particularly to a method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography, belonging to the technical field of pharmaceutical analysis. Technical Background

[0002] Furosemide is a potent diuretic drug, a loop diuretic widely used in the treatment of congestive heart failure and edema, used to treat edema caused by diseases of the heart, liver, kidney, etc., especially in cases where other diuretics are ineffective; it can be used to treat acute pulmonary edema, cerebral edema, acute renal failure and hypertension and other diseases; combined with fluid replacement, this product can promote the excretion of poisons.

[0003] The chemical name of furosemide is: 2-[(2-furfuryl)amino]-5-(sulfamoyl)-4-chlorobenzoic acid, alias furosemide; the chemical structural formula is as follows:

[0004]

[0005] Injection is one of the most widely used and important dosage forms, and its dosage form advantages: rapid and reliable action. Its liquid medicine is directly injected into tissues or blood vessels, without an absorption process or with a very short absorption process. Therefore, the blood concentration can quickly reach the peak and play a role. It does not pass through the digestive tract, has no first-pass effect, and the drug content is not easily lost. Therefore, the curative effect is reliable. Furosemide was first synthesized by Sanofi in 1962 and was launched in the United States on March 20, 1968, under the trade name "LASIX", with the licensee being "SANOFIAVENTISUS", having the status of RLD, and has now been delisted; it was launched in France on May 6, 1988, under the trade name "LASILIX 20mg / 2ml, solution injectable en ampoule", with the licensee "SANOFIAVENTIS FRANCE" and having the status of a reference preparation; it was launched in Japan in June 1965, under the trade name "ラシックス注20mg(LASIX)", with the licensee being "サノフィ株式会社(赛诺菲株式会社)"; the original research product has not been launched in China. The 24th batch of reference preparations for generic drugs announced by the National Medical Products Administration is as shown in Table 1 below:

[0006] Table 1

[0007]

[0008]

[0009] Combined with the structural characteristics of furosemide, furosemide is unstable under light conditions and is prone to degradation to produce furfural and furfuryl alcohol, and this impurity is a possible mutagenic genotoxic impurity.

[0010]

[0011] According to the toxicity data published by CPDB (Carcinogenic Potential Database), as shown in Table 2 below:

[0012] Table 2

[0013]

[0014] According to the domestic and international clinical instructions for use and dosage of furosemide injection, its maximum daily dose is 250 mg / day. The control limits of furfuryl alcohol and furfural in furosemide injection are calculated as follows according to the mutagenic impurity control limits:

[0015]

[0016]

[0017] A patent application was found (patent application number: CN201910734512.3). The high-performance liquid phase detection method for the genotoxic impurity of furosemide disclosed in the patent can only detect the impurity furfural, but cannot detect both furfural and furfuryl alcohol at the same time, and cannot effectively control the genotoxic impurities of furosemide; moreover, the mobile phase aqueous phase of this method is phosphate buffer, the mobile phase system is complex, and the detection cost is increased; the method lacks sufficient basis for formulating the detection control limit of furfural; and no comprehensive methodological research has been conducted on the detection method.

[0018] The published document "Determination and Evaluation of Furfural in Furosemide and Its Tablets" (Wu Jingfang, Chen Minyu, et al. (1. Guangzhou Institute of Drug Control; 2. China Food and Drug Inspection Institute)) discloses a high performance liquid chromatography detection method for determining furfural in furosemide raw materials and tablets. The method has the following disadvantages: it cannot simultaneously detect another genotoxic impurity furfuryl alcohol, and cannot effectively control the genotoxic impurities of furosemide; and the mobile phase aqueous phase of the method is phosphate buffer, the mobile phase system is complex, and the detection cost is increased; the method has insufficient basis for formulating the detection control limit of furfural, and the limit is not calculated according to the actual maximum daily dose in the clinical instructions; the detection limit of furfural under this method is only 4ng / ml.

[0019] The document "Detection of trace genotoxic impurities in furosemide and its preparations" (Zhao Yanan, He Nianping, etc.) has been published. The high-performance liquid phase detection method disclosed in the document determines furfural in furosemide injection, which is to control the genotoxic impurity furfural at the same time as the related substance detection method. Disadvantages of this method: under the conditions of this method, another genotoxic impurity furfuryl alcohol cannot be detected, and the genotoxic impurities of furosemide cannot be effectively controlled; and the mobile phase water phase of this method is phosphate buffer, the mobile phase system is complex, and the detection cost is increased; the method lacks a basis for the formulation of the detection control limit of furfural, and the limit is not calculated according to the actual maximum daily dose of the clinical instructions; the detection limit of furfural under this method is only 4ng / ml.

[0020] Therefore, a method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography was developed, which adopted a dual-wavelength gradient elution method to detect and control two genotoxic impurities under the same chromatographic conditions; the test operation was convenient, the detection sensitivity and detection limit were high, the recovery rate was good, the durability was high, the detection stability and accuracy were high, and solving a technical problem in the field of this variety, saving detection costs, and effectively controlling the quality and safety of the product became a technical problem that urgently needed to be solved in this technical field. Summary of the invention

[0021] The object of the present invention is to provide a method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography, which adopts a dual-wavelength gradient elution method to achieve detection and control of two genotoxic impurities under the same chromatographic conditions; the inspection operation is convenient, the detection sensitivity and detection limit are high, the recovery rate is good, the durability is high, the detection stability and accuracy are high, a technical problem in the field of this variety is solved, the detection cost is saved, and the quality and safety of the product are effectively controlled.

[0022] The above invention object is achieved through the following technical solutions:

[0023] A method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography comprises the following steps:

[0024] (1) HPLC conditions

[0025] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0026] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate solution, pH adjusted to 2.0-3.0 with phosphoric acid;

[0027] Mobile phase B: mobile phase A-acetonitrile [(45:55)~(55:45)];

[0028] Furfuryl alcohol detection wavelength: 225-235nm; Furfural detection wavelength: 268-276nm;

[0029] Flow rate: 0.8-1.2 ml / min; injection volume: 45-55 μl;

[0030] Perform gradient elution;

[0031] (2) Sample preparation

[0032] Solvent: acetonitrile-water = 10:80-30:70;

[0033] Blank excipient solution: Take 5 ml of blank excipient, dilute with solvent to 10 ml, and mix well;

[0034] Test solution: Take an appropriate amount of furosemide injection and dilute it with solvent to make a solution containing 5 mg per 1 ml;

[0035] Reference solution: Take appropriate amount of furfuryl alcohol and furfural, dilute with solvent, and prepare furfuryl alcohol solution with concentration of 0.05 μg / ml and furfural solution with concentration of 1 μg / ml respectively;

[0036] (3) Detection method

[0037] Measure the solvent, blank excipient solution, reference solution, and test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the peak area according to the external standard method.

[0038] Preferably, in step (1), the mobile phase A is a 0.01 mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.5 with phosphoric acid.

[0039] Preferably, in step (1), the mobile phase A-acetonitrile is 50:50.

[0040] Preferably, in step (1), the wavelengths are: 230 nm for detecting furfuryl alcohol; 272 nm for detecting furfural.

[0041] Preferably, in step (1), the flow rate is 1 ml / min.

[0042] Preferably, in step (1), the injection volume is 50 μl.

[0043] Preferably, in step (1), the gradient elution procedure is as shown in Table 3.

[0044] Table 3

[0045]

[0046]

[0047] Preferably, in step (2), solvent: acetonitrile-water = 20:80

[0048] Preferably, in step (3), the blank auxiliary material solution and solvent do not interfere with the detection; and the separation degree between the main component and other impurities and furfuryl alcohol and furfural is ≥1.5.

[0049] Preferably, in step (3), the main component is furosemide; and the other impurities are impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F.

[0050] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0051] The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography of the present invention can realize the simultaneous detection of two mutagenic impurities in one detection method, has strong specificity, high sensitivity, good linear relationship, high accuracy, good detection durability and stability; can accurately reflect the true contents of the two mutagenic impurities furfuryl alcohol and furfural in furosemide injection, effectively reduces the detection cost, effectively improves the quality control of furosemide injection, ensures the quality and quantity safety of the drug, and reduces the clinical toxic and side effects that may be caused by the drug.

[0052] The present invention is further described below through the accompanying drawings and specific embodiments, but it is not intended to limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 HPLC spectrum of the blank solvent in Example 1 of the present invention;

[0054] Figure 2 HPLC spectrum of the blank auxiliary material in Example 1 of the present invention;

[0055] Figure 3 HPLC spectrum of the separation solution in Example 1 of the present invention;

[0056] Figure 4 HPLC spectrum of the test solution in Example 1 of the present invention;

[0057] Figure 5 The HPLC spectrum was not tested with reference to the chromatographic conditions of "Glucosamine Sulfate Capsules, JX20160314". DETAILED DESCRIPTION

[0058] Unless otherwise specified, the materials, reagents, equipment and instruments used in the following examples of the present invention are all conventional products available in the art, and the units described are all weight units.

[0059] Example 1

[0060] A method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography comprises the following steps:

[0061] (1) HPLC conditions

[0062] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0063] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate solution, pH adjusted to 2.5 with phosphoric acid;

[0064] Mobile phase B: mobile phase A-acetonitrile 50:50;

[0065] Furfuryl alcohol detection wavelength: 230nm; Furfural detection wavelength: 272nm;

[0066] Flow rate: 1.0 ml / min; injection volume: 50 μl;

[0067] Perform gradient elution;

[0068] (2) Sample preparation

[0069] Solvent: acetonitrile-water = 20:80;

[0070] Blank excipient solution: Take 5 ml of blank excipient, dilute with solvent to 10 ml, and mix well;

[0071] Test solution: Take an appropriate amount of furosemide injection and dilute it with solvent to make a solution containing 5 mg per 1 ml;

[0072] Reference solution: Take appropriate amount of furfuryl alcohol and furfural, dilute with solvent, and prepare furfuryl alcohol solution with concentration of 0.05 μg / ml and furfural solution with concentration of 1 μg / ml respectively;

[0073] Positioning solution: accurately weigh appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and furosemide reference substance, place in a volumetric bottle, dilute with solvent, and prepare solutions with a concentration of 10 μg / ml as positioning solutions; take an appropriate amount of furfuryl alcohol, dilute with solvent, and prepare a 0.1 μg / ml furfuryl alcohol positioning solution; take an appropriate amount of furfural, dilute with solvent, and prepare a 1 μg / ml furfural positioning solution;

[0074] Separation solution: Take appropriate amounts of impurity A, furosemide reference substance, impurity B, impurity C, impurity D, impurity E, impurity F, furosemide injection, furfural reference substance, and furfuryl alcohol reference substance, respectively, place them in the same volumetric flask, add solvent to dilute, and prepare a mixed solution containing impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F at a concentration of 10 μg / ml, furfuryl alcohol at a concentration of 0.05 μg / ml, and furfural at a concentration of 1 μg / ml, as the separation solution;

[0075] (3) Detection method

[0076] Avoid light, measure the solvent, blank excipient solution, reference solution, and test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the peak area according to the external standard method;

[0077] In step (1), the gradient elution procedure is shown in Table 3.

[0078] Table 3

[0079]

[0080]

[0081] Example 2

[0082] A method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography comprises the following steps:

[0083] (1) HPLC conditions

[0084] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0085] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate solution, pH adjusted to 2.0 with phosphoric acid;

[0086] Mobile phase B: mobile phase A-acetonitrile 45:55;

[0087] Furfuryl alcohol detection wavelength: 225nm; Furfural detection wavelength: 268nm;

[0088] Flow rate: 0.8 ml / min; injection volume: 45 μl;

[0089] Perform gradient elution;

[0090] (2) Sample preparation

[0091] Solvent: acetonitrile-water = 10:90;

[0092] Blank excipient solution: Take 5 ml of blank excipient, dilute with solvent to 10 ml, and mix well;

[0093] Test solution: Take an appropriate amount of furosemide injection and dilute it with solvent to make a solution containing 5 mg per 1 ml;

[0094] Reference solution: Take appropriate amount of furfuryl alcohol and furfural, dilute with solvent, and prepare furfuryl alcohol solution with concentration of 0.05 μg / ml and furfural solution with concentration of 1 μg / ml respectively;

[0095] (3) Detection method

[0096] Avoid light, measure the solvent, blank excipient solution, reference solution, and test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the peak area according to the external standard method;

[0097] In step (1), the gradient elution procedure is shown in Table 3.

[0098] Table 3

[0099]

[0100]

[0101] Example 3

[0102] A method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography comprises the following steps:

[0103] (1) HPLC conditions

[0104] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0105] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate solution, pH adjusted to 3.0 with phosphoric acid;

[0106] Mobile phase B: mobile phase A-acetonitrile 55:45;

[0107] Furfuryl alcohol detection wavelength: 235nm; Furfural detection wavelength: 276nm;

[0108] Flow rate: 1.2 ml / min; injection volume: 55 μl;

[0109] Perform gradient elution;

[0110] (2) Sample preparation

[0111] Solvent: acetonitrile-water = 30:70;

[0112] Blank excipient solution: Take 5 ml of blank excipient, dilute with solvent to 10 ml, and mix well;

[0113] Test solution: Take an appropriate amount of furosemide injection and dilute it with solvent to make a solution containing 5 mg per 1 ml;

[0114] Reference solution: Take appropriate amount of furfuryl alcohol and furfural, dilute with solvent, and prepare furfuryl alcohol solution with concentration of 0.05 μg / ml and furfural solution with concentration of 1 μg / ml respectively;

[0115] (3) Detection method

[0116] Avoid light, measure the solvent, blank excipient solution, reference solution, and test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the peak area according to the external standard method;

[0117] In step (1), the gradient elution procedure is shown in Table 3.

[0118] Table 3

[0119]

[0120] Compared with the existing detection technology of mutagenic genotoxic impurities of furosemide injection, the present invention has the following advantages:

[0121] (1) The chromatographic conditions of "Glucosamine Sulfate Capsules, JX20160314" were used for testing. The results showed that the solvent peak interfered with the furfuryl alcohol peak (see Figure 5 ), based on this method, the method was optimized and improved. At the respective detection wavelengths of furfural, the separation between each impurity and the main component and furfural was good, which did not affect the detection of furfural; it can be judged that the specificity of this method is good (see Figure 3 ).

[0122] Table 4 (Experimental results of Example 1)

[0123]

[0124] (2) Through ultraviolet spectral scanning of the two mutagenic impurities in furosemide injection, it was found that the ultraviolet maximum absorption wavelengths of the two impurities were different. In the process of chromatographic condition optimization, dual-wavelength detection was developed under the same chromatographic conditions, which enabled the simultaneous detection of two impurities under the same chromatographic detection conditions, saving the detection cost of traditional methods and filling the gaps in existing detection technologies.

[0125] (3) The detection method of the present invention has been verified by a complete methodology, has strong specificity, and the blank solvent and blank auxiliary materials do not interfere with the detection of two mutagenic impurities (furfural and furfuryl alcohol); the separation between the two chromatographic peaks of furfural and furfuryl alcohol meets the requirements and is greater than 1.5; the detection sensitivity is high, and the detection limit of furfural in the present invention is 0.04 ng, and the detection limit of furfuryl alcohol is 0.38 ng, and the detection sensitivity is higher than that of the existing detection technology method; the detection accuracy is high, and the recovery test results of furfural and furfuryl alcohol show that the recovery test results at three concentrations of 80%, 100% and 120% meet the requirements, and the recovery rate of furfural is 90.1%, and the recovery rate of furfuryl alcohol is 100.8%; the detection method is stable, and the durability test results of the detection method show that the chromatographic column, column temperature, pH value of the buffer salt, flow rate, etc. in the fine-tuning of the chromatographic conditions do not affect the detection results, and the detection method of the present invention has good durability.

[0126] Compared with the existing mutagenicity detection technology for furosemide injection, the detection method of the present invention can effectively, accurately and stably detect the contents of two mutagenic impurities, furfural and furfuryl alcohol, in furosemide injection, breaking through the existing detection technology and realizing the control of two mutagenic impurities by the detection box under the same high performance liquid phase detection chromatographic conditions, saving detection costs and more effectively improving the quality and safety of clinical drugs.

[0127] The above description is only a preferred specific implementation mode of the present invention and is not intended to limit the scope of protection of the present invention. Any person skilled in the art may make various changes and modifications according to the technical solution of the present invention without departing from the spirit and scope of the present invention, which still fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography, comprising the following steps: (1) HPLC conditions Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate solution, pH adjusted to 2.0-3.0 with phosphoric acid; Mobile phase B: mobile phase A-acetonitrile [(45:55)~(55:45)]; Furfuryl alcohol detection wavelength: 225-235nm; Furfural detection wavelength: 268-276nm; Flow rate: 0.8-1.2 ml / min; injection volume: 45-55 μl; Perform gradient elution; (2) Sample preparation Solvent: acetonitrile-water = 10:90-30:70; Blank excipient solution: Take 5 ml of blank excipient, dilute with solvent to 10 ml, and mix well; Test solution: Take an appropriate amount of furosemide injection and dilute it with solvent to make a solution containing 5 mg per 1 ml; Reference solution: Take appropriate amount of furfuryl alcohol and furfural, dilute with solvent, and prepare furfuryl alcohol solution with concentration of 0.05 μg / ml and furfural solution with concentration of 1 μg / ml respectively; (3) Detection method Measure the solvent, blank excipient solution, reference solution, and test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the peak area according to the external standard method.

2. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 1, characterized in that: In step (1), the mobile phase A is a 0.01 mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.5 with phosphoric acid.

3. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 1, characterized in that: In step (1), the mobile phase A-acetonitrile is 50:

50.

4. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 1, characterized in that: In step (1), the wavelengths are: 230 nm for detecting furfuryl alcohol and 272 nm for detecting furfural.

5. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 1, characterized in that: In step (1), the flow rate is 1 ml / min.

6. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 1, characterized in that: In step (1), the injection volume is 50 μl.

7. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 1, characterized in that: In step (1), the gradient program is as follows.

8. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 1, characterized in that: In step (3), the blank excipient solution and solvent do not interfere with the detection.

9. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 1, characterized in that: In step (3), the separation degree between the main component and other impurities and furfuryl alcohol and furfural is greater than 1.

5.

10. The method for detecting mutagenic impurities in furosemide injection by high performance liquid chromatography according to claim 9, characterized in that: In step (3), the main component is furosemide; and the other impurities are impurity A, impurity B, impurity C, impurity D, impurity E and impurity F.

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