Method for detecting n-butyraldehyde in bumetanib

Abstract

The invention provides a method for detecting n-butyraldehyde in bumetanib, which adopts a gas chromatography detection method, and the chromatographic conditions are as follows: a chromatographic column is a DB-1 quartz capillary column, and the specification is 30m * 0.32 mm * 5 [mu] m; the stationary liquid is 100% dimethyl polysiloxane; the initial column temperature is 35-45 DEG C; the temperature of a sample inlet is 200 DEG C; the sample size is 1ml; the split ratio is 10: 1; the carrier gas is nitrogen, and the flow velocity is 0.8-1.2 ml / min; a detector is a hydrogen flame ionization detector, and the temperature of the detector is 250 DEG C; the headspace balance temperature is 85 DEG C, and the balance time is 30 minutes. According to the method, the n-butyraldehyde in the bumetanib is successfully separated by selecting proper gas chromatography conditions, the peak pattern of the n-butyraldehyde is relatively good, the n-butyraldehyde is not interfered by the bumetanib and other impurities in the bumetanib, and the content of the n-butyraldehyde is accurately detected; based on the method, the residual risk of n-butyraldehyde impurities can be effectively controlled, and the medicine safety is guaranteed.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis and detection, and in particular to a method for detecting n-butyraldehyde in bumetanide. Background Art

[0002] Bumetanide, commonly known as furosemide, has a chemical name of 3-n-butylamino-4-phenoxy-5-sulfamylbenzoic acid. It is a new type of non-benzothiazide diuretic that does not contain mercury and is highly effective and low in toxicity. Its diuretic effect is 40 times higher than that of furosemide. It is used to treat hepatitis B edema. The oral dose is 1 to 2 mg / d, which is 1 / 40 of that of furosemide.

[0003] Patent document CN101591276A discloses a method for synthesizing bumetanide. The method uses 3-amino-4-phenoxy-5-sulfonamidobenzoic acid and n-butyraldehyde as raw materials, in the presence of trihalogenated boron etherate, to prepare 3-butylimine-4-phenoxy-5-sulfonamidobenzoic acid, and then uses palladium-carbon to catalyze the hydrogenation of 3-butylimine-4-phenoxy-5-sulfonamidobenzoic acid to obtain the bumetanide product.

[0004] Patent WO2012154728A1 also discloses a method for synthesizing bumetanide, which is to use 3-amino-4-phenoxy-5-sulfonamide benzoic acid and deuterated n-butyraldehyde as raw materials, and then react with deuterated hydrogen / Pd / C or NaCNBD3 to prepare the corresponding product.

[0005] At present, the process of synthesizing bumetanide basically uses n-butyraldehyde as the raw material, so n-butyraldehyde may remain in the generated bumetanide. According to the ICHM7 (Evaluation and Control of Mutagenic Impurities) guidelines, n-butyraldehyde is a genotoxic warning structure and has potential safety risks. Therefore, it is of great significance to control the content of n-butyraldehyde in bumetanide.

[0006] However, the detection method of butyraldehyde is not included in the domestic and international pharmacopoeia standards for bumetanide. In the absence of an effective detection method, its residual risk cannot be controlled. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting butyraldehyde in bumetanide. Based on this method, the residual risk of butyraldehyde impurities can be effectively controlled to ensure the safety of the drug.

[0008] The structural information of bumetanide and n-butyraldehyde impurities of the present invention is shown in Table 1.

[0009] Table 1 Structural information of bumetanide and n-butyraldehyde impurities

[0010]

[0011] The present invention adopts the following technical solutions to solve the above technical problems:

[0012] A method for detecting n-butyraldehyde in bumetanide, using gas chromatography detection, the chromatographic conditions are:

[0013] The chromatographic column was a DB-1 quartz capillary column with a specification of 30m×0.32mm×5μm; the stationary liquid was 100% dimethylpolysiloxane; the initial column temperature was 35-45°C;

[0014] Injection port temperature 200℃; injection volume 1ml; split ratio 10:1; carrier gas is nitrogen, nitrogen flow rate (column flow) 0.8~1.2ml / min;

[0015] The detector is a hydrogen flame ionization detector with a detector temperature of 250°C;

[0016] The headspace equilibrium temperature was 85°C and the equilibrium time was 30 min.

[0017] As one of the preferred embodiments of the present invention, the chromatographic conditions further include using programmed temperature rise: the starting temperature is 35-45°C, maintained for 5 minutes, and then the temperature is increased to 200°C at a rate of 10°C per minute and maintained for 5 minutes.

[0018] As one of the preferred embodiments of the present invention, the method for detecting n-butyraldehyde in bumetanide comprises the following specific steps:

[0019] (1) Solution preparation

[0020] Diluent: organic solvent;

[0021] Test solution: Prepare bumetanide raw material by dissolving it in diluent;

[0022] Reference solution: prepare n-butyraldehyde by dissolving it in a diluent;

[0023] (2) Gas chromatography analysis

[0024] The gas chromatograph was used for detection. The gas chromatographic conditions were as follows: the chromatographic column was a DB-1 quartz capillary column with a specification of 30m×0.32mm×5μm; the stationary liquid was 100% dimethylpolysiloxane; the initial column temperature was 35-45°C; the temperature was programmed; the injection port temperature was 200°C; the injection volume was 1ml; the split ratio was 10:1; the carrier gas was nitrogen with a nitrogen flow rate of 0.8-1.2ml / min; the detector was a hydrogen flame ionization detector with a detector temperature of 250°C; the headspace equilibrium temperature was 85°C and the equilibrium time was 30min;

[0025] Take the test solution and the reference solution, inject them into the headspace respectively, record the chromatograms, and calculate the content of each component.

[0026] As one of the preferred embodiments of the present invention, in the step (1), the diluent is specifically 80% dimethyl sulfoxide, which is used to dilute and prepare the test solution and the reference solution.

[0027] As one of the preferred embodiments of the present invention, in step (1), the concentration of the test solution is 100 mg / ml (bumetanide).

[0028] As one of the preferred embodiments of the present invention, in step (1), the concentration of the reference solution is 50 μg / ml (n-butyraldehyde).

[0029] As one of the preferred embodiments of the present invention, in step (2), the gas chromatograph is a Shimadzu GC-2030 gas chromatograph from Japan.

[0030] As one of the preferred embodiments of the present invention, in step (2), the content of each component is calculated according to the external standard method.

[0031] The advantages of the present invention compared to the prior art are:

[0032] (1) The present invention successfully separates the n-butyraldehyde impurity in the bumetanide product by selecting appropriate gas chromatography conditions, and can achieve a good n-butyraldehyde peak shape without interference from bumetanide and other impurities in bumetanide, thereby accurately detecting the n-butyraldehyde content; based on this method, the present invention can effectively control the residual risk of n-butyraldehyde impurities and ensure the safety of bumetanide drugs;

[0033] (2) The detection method provided by the present invention has strong specificity and good durability. Changing the chromatographic conditions such as column flow rate and initial column temperature within a small range will not affect the detection of n-butyraldehyde. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the chromatogram of the blank solvent, reference solution, test solution and separation test solution in Example 1;

[0035] Figure 2 is the linear relationship diagram of n-butyraldehyde in Experimental Example 1;

[0036] Figure 3 It is the chromatogram under different initial column temperature and column flow conditions in Experimental Example 1. DETAILED DESCRIPTION

[0037] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the raw materials, reagents, equipment and methods used in the following embodiments are conventional raw materials, reagents, equipment and methods in the art unless otherwise specified, and will not be repeated.

[0038] Example 1

[0039] A method for detecting n-butyraldehyde in bumetanide of the present embodiment adopts gas chromatography detection method, comprising the following steps:

[0040] (1) Solution preparation

[0041] Blank solvent: 80% dimethyl sulfoxide.

[0042] Test solution: Take a bumetanide sample, accurately weigh it, and dilute it with 80% dimethyl sulfoxide solution to make a solution containing 100 mg of n-butyraldehyde per 1 ml.

[0043] Reference solution: Take an appropriate amount of n-butyraldehyde, weigh it accurately, and dilute it with 80% dimethyl sulfoxide solution to make a solution containing 50 μg of n-butyraldehyde per 1 ml.

[0044] Separation test solution: Take 0.5 g of bumetanide sample, weigh it accurately, place it in an empty bottle, accurately add 5 ml of reference solution, shake well, seal, and obtain.

[0045] (2) Gas chromatography analysis

[0046] The gas chromatograph (Shimadzu, Japan, GC-2030) was used for detection. The gas chromatographic conditions were as follows:

[0047] The chromatographic column is a DB-1 quartz capillary column with specifications of 30m×0.32mm×5μm; the stationary liquid is 100% dimethylpolysiloxane; the initial column temperature is 40℃, maintained for 5min, and then increased to 200℃ at a rate of 10℃ per min, and maintained for 5min; the injection port temperature is 200℃; the injection volume is 1ml; the split ratio is 10:1; the carrier gas is nitrogen, and the nitrogen flow rate (column flow) is 1.0ml / min; the detector is a hydrogen flame ionization detector, and the detector temperature is 250℃; the headspace equilibrium temperature is 85℃, and the equilibrium time is 30min.

[0048] Take blank solvent, reference solution, test solution and separation test solution respectively, inject samples under the above chromatographic conditions, record the chromatogram, if there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the n-butyraldehyde peak, calculate the peak area according to the external standard method.

[0049] The chromatograms of the blank solvent, reference solution, test solution and separation test solution of this example are shown in Figure 1 .

[0050] Example 2

[0051] The method for detecting n-butyraldehyde in bumetanide in this embodiment is basically the same as that in Example 1, except that the initial column temperature is 35° C. in the gas chromatography conditions.

[0052] Example 3

[0053] The method for detecting n-butyraldehyde in bumetanide in this embodiment is basically the same as that in Example 1, except that the initial column temperature is 45° C. in the gas chromatography conditions.

[0054] Example 4

[0055] The method for detecting n-butyraldehyde in bumetanide in this embodiment is basically the same as that in Example 1, except that the nitrogen flow rate (column flow rate) is 0.8 ml / min.

[0056] Example 5

[0057] The method for detecting n-butyraldehyde in bumetanide in this embodiment is basically the same as that in Example 1, except that the nitrogen flow rate (column flow rate) is 1.2 ml / min.

[0058] Experimental Example 1

[0059] This experimental example is a methodological verification of the method of the present invention.

[0060] 1. Exclusivity

[0061] The instrument and chromatographic conditions were the same as those in Example 1.

[0062] Investigate the separation between the peaks.

[0063] The blank solvent, test solution, reference solution and separation test solution are the same as those in Example 1.

[0064] Butyraldehyde positioning solution: Take 20 μl of n-butyraldehyde and place it in a headspace bottle to which 5 ml of 80% dimethyl sulfoxide solution has been added. Shake well and seal.

[0065] Methanol positioning solution: Take 20 μl of methanol and place it in a headspace bottle to which 5 ml of 80% dimethyl sulfoxide solution has been added, shake well, and seal.

[0066] Benzene positioning solution: Take 20 μl of benzene and place it in a headspace bottle to which 5 ml of 80% dimethyl sulfoxide solution has been added. Shake well and seal.

[0067] The blank solvent, test solution, reference solution, resolution test solution and each positioning solution were injected into the headspace respectively. The resolution results are shown in Table 2.

[0068] Table 2 Specificity test results

[0069]

[0070] The results showed that the blank solvent (80% dimethyl sulfoxide solution) had no interference with the components to be tested; the test solvents methanol and benzene had no interference with the detection; in the separation test solution, the separation between the n-butyraldehyde peak and the adjacent peaks was greater than 1.5.

[0071] 2. Limit of Detection and Limit of Quantitation

[0072] The blank solvent was the same as in Example 1.

[0073] Take an appropriate amount of n-butyraldehyde, weigh it accurately, dilute it with 80% dimethyl sulfoxide solution at a certain ratio to make a solution of appropriate concentration. When the signal-to-noise ratio (S / N) is 3, prepare the detection limit solution; when the signal-to-noise ratio (S / N) is about 10, prepare the quantification limit solution. Prepare 6 portions in the same way.

[0074] Take the blank solvent, detection limit solution and quantitative limit solution, inject them respectively, and record the chromatograms. The results are shown in Table 3 and Table 4.

[0075] Table 3 Detection limit test results

[0076] name Concentration (μg / ml) Peak area Signal-to-Noise Ratio (S / N) Equivalent sample concentration (%) Butyraldehyde 0.0336 353 3.62 0.00003

[0077] Table 4 Quantitation limit test results

[0078]

[0079] The results showed that the detection limit concentration was equivalent to 0.00003% of the sample concentration, and the detection sensitivity met the limit requirement; after repeated injection of 6 injections of the quantitative limit solution, the RSD of the peak area of ​​n-butyraldehyde was ≤10.0%, which met the quantitative limit precision requirement.

[0080] 3. Linear

[0081] The blank solvent was the same as in Example 1.

[0082] Reference substance stock solution: Take an appropriate amount of n-butyraldehyde, weigh it accurately, add 80% dimethyl sulfoxide solution to dilute it to make a solution containing about 1 mg of n-butyraldehyde per 1 ml, shake well, and the solution is ready.

[0083] Linear solution ①: Accurately pipette 5 ml of the quantitative limit solution under the above detection limit and quantitative limit items, place it in an empty bottle, seal it, and you have it.

[0084] Linear solution ②: 50% solution. Accurately pipette 0.5 ml of the above reference stock solution into a 20 ml volumetric flask, dilute to scale with 80% dimethyl sulfoxide solution, shake well; accurately pipette 5 ml into an empty bottle, seal, and you have it.

[0085] Linear solution ③: 80% solution. Accurately pipette 0.4 ml of the above reference stock solution into a 10 ml volumetric flask, dilute to scale with 80% dimethyl sulfoxide solution, shake well; accurately pipette 5 ml into an empty bottle, seal, and you have it.

[0086] Linear solution ④: 100% solution. Accurately pipette 0.5 ml of the above reference stock solution into a 10 ml volumetric flask, dilute to scale with 80% dimethyl sulfoxide solution, shake well; accurately pipette 5 ml into an empty bottle, seal, and you have it.

[0087] Linear solution ⑤: 150% solution. Accurately pipette 0.75 ml of the above reference stock solution into a 10 ml volumetric flask, dilute to scale with 80% dimethyl sulfoxide solution, shake well; accurately pipette 5 ml into an empty bottle, seal, and you have it.

[0088] Take the blank solvent and each concentration linear solution and inject them into the headspace. The results are shown in Table 5 and Figure 2 .

[0089] Table 5 Linearity test results

[0090]

[0091] The results showed that the linear relationship of n-butyraldehyde was good in the concentration range of 0.13μg / ml to 75.20μg / ml.

[0092] 4. Accuracy

[0093] The blank solvent, test solution and reference solution were the same as those in Example 1.

[0094] Reference substance stock solution: Take an appropriate amount of n-butyraldehyde, weigh it accurately, add 80% dimethyl sulfoxide solution to dilute it to make a solution containing about 2.5 mg of n-butyraldehyde per 1 ml, shake well, and the solution is ready.

[0095] 80% accuracy dilution solution: Accurately pipette 0.8 ml of the above reference substance stock solution into a 50 ml volumetric flask, dilute to the scale with 80% dimethyl sulfoxide solution, mix well, and the solution is ready.

[0096] 120% accuracy dilution: Accurately pipette 1.2 ml of the above reference substance stock solution into a 50 ml volumetric flask, dilute to the scale with 80% dimethyl sulfoxide solution, mix well, and the solution is ready.

[0097] 80% accuracy solution: Take about 0.5g of bumetanide sample, weigh accurately, place in an empty bottle, accurately add 5ml of the above 80% accuracy diluent, seal, and dissolve by ultrasonic. Prepare 3 portions in the same way.

[0098] 100% accuracy solution: Take about 0.5g of bumetanide sample, weigh accurately, place in an empty bottle, accurately add 5ml of the above reference solution, seal, and dissolve by ultrasonic. Prepare 3 portions in the same way.

[0099] 120% accuracy solution: Take about 0.5g of bumetanide sample, weigh accurately, place in an empty bottle, accurately add 5ml of the above 120% accuracy diluent, seal, and dissolve by ultrasonic. Prepare 3 portions in the same way.

[0100] Take the blank solvent, reference solution and each accuracy solution and inject them into the headspace. The results are shown in Table 6.

[0101] Table 6 Summary of n-butyraldehyde accuracy test data

[0102]

[0103] The results showed that the recovery rate of n-butyraldehyde was between 84.78% and 89.16%, and the recovery rate RSD was ≤10.0%, which met the requirements of method validation. The method had good accuracy in determining n-butyraldehyde.

[0104] 5. Durability

[0105] The blank solvent and reference solution were the same as in Example 1.

[0106] Durability solution: Accurately measure 5 ml of the above reference solution, shake well, seal, and prepare 10 portions in the same way.

[0107] Adjust the chromatographic conditions according to Table 7, and take the blank solvent and durability solution for headspace injection respectively. Figure 3 and Table 8, Table 9.

[0108] Table 7 Parameters of chromatographic condition changes

[0109] Chromatographic parameters Specified value Range of change Initial column temperature (℃) 40 35、45 Column flow rate (ml / min) 1.0 0.8、1.2

[0110] Table 8 Test results of different initial column temperatures

[0111]

[0112] Table 9 Test results of different column flow rates

[0113]

[0114]

[0115] Combination Figure 1 as well as Figure 3 The results of Table 8 and Table 9 show that: (1) With the increase of the initial column temperature, the retention time of the n-butyraldehyde peak is gradually advanced, the separation between the n-butyraldehyde peak and the adjacent peaks is greater than 1.5, and the peak area of ​​the n-butyraldehyde peak has no significant change; it can be seen that changing the initial temperature of the column oven has no significant effect on the test results; (2) With the increase of the flow rate of the chromatographic column, the retention time of the n-butyraldehyde peak is gradually advanced, the separation between the n-butyraldehyde peak and the adjacent peaks is greater than 1.5, and the peak area of ​​the n-butyraldehyde peak has no significant change; it can be seen that changing the flow rate of the chromatographic column has no significant effect on the test results. Therefore, this method has good durability.

[0116] In summary, the detection method provided by the present invention has strong specificity and good durability. Changing chromatographic conditions such as column flow rate and initial column temperature within a small range will not affect the detection of n-butyraldehyde; the method provided by the present invention can accurately detect the n-butyraldehyde impurity in bumetanide, and the separation degree between bumetanide and each impurity in bumetanide and n-butyraldehyde is good. The detection results are accurate and reliable, and the quality of bumetanide can be controlled more scientifically.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting n-butyraldehyde in bumetanide, characterized in that: The gas chromatography detection method was adopted, and the chromatographic conditions were: The chromatographic column was a DB-1 quartz capillary column with a specification of 30m×0.32mm×5μm; the stationary liquid was 100% dimethylpolysiloxane; the initial column temperature was 35-45°C; Injection port temperature 200℃; injection volume 1ml; split ratio 10:1; carrier gas is nitrogen, nitrogen flow rate 0.8~1.2ml / min; The detector is a hydrogen flame ionization detector, and the detector temperature is 250°C; The headspace equilibrium temperature was 85°C and the equilibrium time was 30 min.

2. The method for detecting n-butyraldehyde in bumetanide according to claim 1, characterized in that The chromatographic conditions also include using programmed temperature increase: the starting temperature is 35-45°C, maintained for 5 minutes, and then increased to 200°C at a rate of 10°C per minute, and maintained for 5 minutes.

3. The method for detecting n-butyraldehyde in bumetanide according to claim 1 or 2, characterized in that: The specific steps include: (1) Solution preparation Diluent: organic solvent; Test solution: Prepare bumetanide raw material by dissolving it in diluent; Reference solution: prepare n-butyraldehyde by dissolving it in a diluent; (2) Gas chromatography analysis The gas chromatograph was used for detection. The gas chromatographic conditions were as follows: the chromatographic column was a DB-1 quartz capillary column with a specification of 30m×0.32mm×5μm; the stationary liquid was 100% dimethylpolysiloxane; the initial column temperature was 35-45°C; Program temperature rise; injection port temperature 200℃; injection volume 1ml; split ratio 10:1; carrier gas is nitrogen, nitrogen flow rate 0.8~1.2ml / min; detector is hydrogen flame ionization detector, detector temperature 250℃; headspace equilibrium temperature 85℃, equilibrium time 30min; Take the test solution and the reference solution, inject them into the headspace respectively, record the chromatograms, and calculate the content of each component.

4. The method for detecting n-butyraldehyde in bumetanide according to claim 3, characterized in that: In the step (1), the diluent is specifically 80% dimethyl sulfoxide, which is used to dilute and prepare the test solution and the reference solution.

5. The method for detecting n-butyraldehyde in bumetanide according to claim 3, characterized in that: In the step (1), the concentration of the test solution is 100 mg / ml.

6. The method for detecting n-butyraldehyde in bumetanide according to claim 3, characterized in that: In the step (1), the concentration of the reference solution is 50 μg / ml.

7. The method for detecting n-butyraldehyde in bumetanide according to claim 3, characterized in that: In the step (2), the gas chromatograph is a Shimadzu GC-2030 gas chromatograph from Japan.

8. The method for detecting n-butyraldehyde in bumetanide according to claim 3, characterized in that: In the step (2), the content of each component is calculated according to the external standard method.

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