Method for determining nitrosamine impurity N-nitrosobumetanib in bumetanib

Abstract

The invention discloses a method for determining nitrosamine impurity N-nitrosobumetanib in bumetanib, which comprises the following steps: using a high performance liquid chromatograph and an ultraviolet detector, using octadecyl silane bonded silica gel as a chromatographic column filler, using formic acid solution as a mobile phase A, using methanol as a mobile phase B, the content of the nitrosamine impurity N-nitrosobumetanib in the bumetanib is determined through gradient elution. The novel high performance liquid chromatography is designed, the content of the nitrosamine impurity N-nitrosobumetanib in the bumetanib can be accurately measured, the blank that a method for measuring the N-nitrosobumetanib in the bumetanib bulk drug does not exist at present is filled, and the analysis method is verified; the method for determining the nitrosamine impurity N-nitrosobumetanib in the bumetanib, provided by the invention, is good in repeatability, high in selectivity, good in accuracy and high in precision and sensitivity; according to the invention, the quality control of the medicine can be effectively enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for determining nitrosamine impurity N-nitrosobumetanide in bumetanide. Background Art

[0002] Bumetanide is a powerful diuretic with a diuretic effect 40 to 60 times that of furosemide. It has a rapid onset of action and a good diuretic effect. Compared with furosemide, it has the characteristics of high efficiency, rapid effect, short effect and low toxicity.

[0003] The chemical name of Bumetanide is 3-butylamino-4-phenoxy-5-sulfamoylbenzoic acid, and its molecular formula is C 17 H 20 N2O5S, the structural formula is:

[0004]

[0005] According to the Technical Guidelines for the Study of Nitrosamine Impurities in Chemical Drugs (Trial Implementation), under certain conditions, amine compounds, especially secondary amines, are prone to react with sodium nitrite (NaNO2) or other nitrosating agents to produce nitrosamine impurities. Nitrosamine impurities are a class of compounds with a high risk of carcinogenesis, posing a threat to human health. Therefore, it is necessary to conduct a risk assessment of nitrosamine impurities in raw materials so that the risks of all nitrosamine impurities that may be involved in the actual process can be clearly identified and controlled, thereby achieving an effective way to achieve regulatory expectations.

[0006] After risk assessment, secondary amines are present in both crude and refined products of bumetanide. Although sodium nitrite and other nitrosating agents have been avoided in the synthesis process, a large amount of purified water is used. Nitrite exists in the purified water, which poses a risk of producing nitrosamine impurities. According to the nitrosamine impurity limit data published by the FDA: the PED of N-nitroso-bumetanide is 1.5μg / day. The oral preparation of bumetanide has a specification of 1mg / tablet, and the maximum daily dose is 20mg; injection: the intravenous injection concentration is 0.1mg / ml, the intramuscular injection concentration is 0.25~0.5mg / ml, and the maximum daily dose is 10mg. Therefore, the control limit is set as: 1.5μg / day÷20mg / day×10 6 ppm=75ppm.

[0007] At present, there is no report on the determination method of the nitrosamine impurity N-nitrosobumetanide in the bumetanide raw material drug, and the present invention is specially proposed to achieve stricter quality control of the drug. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide a method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide. The present invention designs a new high performance liquid chromatography method, which can accurately determine the content of the nitrosamine impurity N-nitrosobumetanide in bumetanide, fills the gap of the current lack of a method for determining N-nitrosobumetanide in bumetanide raw materials, and also verifies the analytical method; the method for determining nitrosamine impurities in bumetanide proposed by the present invention has good repeatability, strong selectivity, good accuracy, high precision and high sensitivity; the present invention can effectively enhance the quality control of drugs.

[0009] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0010] A method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide, using a high performance liquid chromatograph, an ultraviolet detector, a chromatographic column filler of octadecylsilane bonded silica gel, a formic acid solution as mobile phase A, methanol as mobile phase B, and gradient elution to determine the content of the nitrosamine impurity N-nitrosobumetanide in bumetanide;

[0011] The bumetanide is 3-butylamino-4-phenoxy-5-sulfamoylbenzoic acid, and its chemical structure is as follows:

[0012]

[0013] The chemical structural formula of the N-nitroso bumetanide is shown in Formula I:

[0014]

[0015] Preferably, the method for determining the nitrosamine impurity N-nitroso-bumetanide in bumetanide comprises the following steps:

[0016] (1) Preparation of reference solution: Take the nitrosamine impurity N-nitrosobumetanide reference substance and add a diluent to prepare a reference solution containing approximately 0.3 μg of N-nitrosobumetanide per 1 ml;

[0017] (2) Preparation of sample solution: Take bumetanide API and add diluent to prepare a sample solution with a concentration of about 4 mg / ml;

[0018] (3) Chromatographic conditions: A high performance liquid chromatograph was used with an ultraviolet detector. The chromatographic column packing was octadecylsilane bonded silica gel. Mobile phase A was formic acid solution, mobile phase B was methanol, and gradient elution was performed. The gradient elution parameters were set as follows:

[0019] From 0 to 20 min, the volume proportion of mobile phase A changed from 70% to 20%, and the volume proportion of mobile phase B changed from 30% to 80%;

[0020] 20-25 min, mobile phase A volume ratio is 20%, mobile phase B volume ratio is 80%;

[0021] From 25 to 25.1 min, the volume proportion of mobile phase A changed from 20% to 70%, and the volume proportion of mobile phase B changed from 80% to 30%;

[0022] 25.1~30min, the volume proportion of mobile phase A is 70%, and the volume proportion of mobile phase B is 30%;

[0023] Accurately measure the reference solution and sample solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the content of the nitrosamine impurity N-nitrosobumetanide in bumetanide using the impurity external standard method;

[0024] (4) Calculation method

[0025] The impurity content is calculated according to the following formula:

[0026]

[0027] A i = peak area of ​​impurities in sample solution;

[0028] A STD =5 for the average peak area of ​​N-nitrosobumetanide in the reference solution;

[0029] m STD = the weight of the impurity reference substance, mg;

[0030] m Spl = sample weight, mg;

[0031] V1 = dilution volume of impurity reference substance, ml;

[0032] V2 = dilution volume of sample, ml;

[0033] P = reference substance content.

[0034] Preferably, in step (1) and step (2), the ratio of the diluent is NMP-water (60:40); more preferably, in the dilution step, 60% volume of NMP should be added first to fully dissolve, and an appropriate amount of water should be added to the scale line, and the mixture should be placed in an ice water bath and quickly cooled to room temperature, and then the volume should be adjusted with water and shaken well.

[0035] Preferably, in step (3), the detection wavelength of the ultraviolet detector is 258-262 nm, more preferably 260 nm.

[0036] Preferably, in step (3), the chromatographic column is Agilent ZORBAX Eclipse Plus C18, with specifications of 4.6 mm×150 mm and 3.5 μm.

[0037] Preferably, in step (3), the column temperature of the chromatographic column is 30-40°C, more preferably 35°C.

[0038] Preferably, in step (3), the mobile phase A is 0.1% formic acid solution.

[0039] Preferably, in step (3), the flow rate of the gradient elution is 0.9-1.1 ml / min, more preferably 1.0 ml / min.

[0040] Preferably, in step (3), the gradient elution parameters are set to:

[0041] 0min, the volume proportion of mobile phase A is 70%, and the volume proportion of mobile phase B is 30%;

[0042] 20 min, mobile phase A volume ratio is 20%, mobile phase B volume ratio is 80%;

[0043] 25 min, mobile phase A volume ratio is 20%, mobile phase B volume ratio is 80%;

[0044] 25.1min, mobile phase A volume ratio is 70%, mobile phase B volume ratio is 30%;

[0045] 30min, the volume ratio of mobile phase A is 70%, and the volume ratio of mobile phase B is 30%.

[0046] Preferably, in step (3), 50 μl of each of the reference solution and the sample solution are accurately measured and injected.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention fills the gap that there is no method for determining the nitrosamine impurity N-nitroso bumetanide in the bumetanide raw material medicine at present; the present invention designs a new high performance liquid chromatography method, which can accurately determine the content of the nitrosamine impurity N-nitroso bumetanide in bumetanide, and also verifies the analytical method; the method of the present invention has good repeatability, strong selectivity, good accuracy, high precision and sensitivity, and can provide reliable data for the quality control of bumetanide. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is the nitrosamine impurity N-nitrosobumetanide in bumetanide of the present invention 1 H-NMR spectrum;

[0050] Figure 2 is the nitrosamine impurity N-nitrosobumetanide in bumetanide of the present invention 13 C-NMR spectrum;

[0051] Figure 3 is the mass spectrum of the nitrosamine impurity N-nitrosobumetanide in bumetanide of the present invention;

[0052] Figure 4 is a chromatogram of the diluent of the present invention;

[0053] Figure 5 is a chromatogram of the reference solution of the present invention, wherein the peak retention time of N-nitroso bumetanide is 14.555 min;

[0054] Figure 6 is a chromatogram of the sample solution of the present invention;

[0055] Figure 7 is a chromatogram of the spiked sample solution of the present invention, wherein the peak retention time of N-nitrosobumetanide is 14.539 min;

[0056] Figure 8 is the chromatogram of the quantitative limit solution of the present invention;

[0057] Fig. 9 It is the chromatogram of the detection limit solution of the present invention. DETAILED DESCRIPTION

[0058] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are only used to further illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art may make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0059] Example 1

[0060] A method for determining nitrosamine impurity N-nitrosobumetanide in bumetanide comprises the following steps:

[0061] (1) Preparation of reference solution: accurately weigh about 3 mg of N-nitroso-bumetanide reference substance, a nitrosamine impurity, and place it in a 20 ml volumetric flask, dissolve it with NMP and dilute it to the mark, and shake well; accurately measure 2 ml and place it in a 100 ml volumetric flask, dilute it to the mark with NMP, and shake well; accurately measure 2 ml and place it in a 20 ml volumetric flask, first add 10 ml of NMP, then add water to the mark, place it in an ice water bath and quickly cool it to room temperature, then dilute it to the mark with water, and shake well to prepare a solution containing 0.3 μg N-nitroso-bumetanide per 1 ml;

[0062] (2) Preparation of sample solution: Take about 80 mg of bumetanide raw material 3-butylamino-4-phenoxy-5-sulfamoylbenzoic acid, accurately weigh it, place it in a 20 ml volumetric flask, first add 12 ml of NMP to dissolve it, then add water to the scale line, quickly cool it to room temperature in an ice water bath, then dilute it with water to the scale, and shake it well;

[0063] (3) Chromatographic conditions: A high performance liquid chromatograph Agilent 1260 (VWD) was used, with a UV detector and an Agilent ZORBAX Eclipse Plus C18 column (4.6 mm × 150 mm, 3.5 μm). Mobile phase A was 0.1% formic acid solution, mobile phase B was methanol, and gradient elution was performed; flow rate was 1.0 ml / min, column temperature was 35°C, wavelength was 260 nm, and injection volume was 50 μl; the gradient elution parameters were set as follows:

[0064] 0min, the volume proportion of mobile phase A is 70%, and the volume proportion of mobile phase B is 30%;

[0065] 20 min, mobile phase A volume ratio is 20%, mobile phase B volume ratio is 80%;

[0066] 25 min, mobile phase A volume ratio is 20%, mobile phase B volume ratio is 80%;

[0067] 25.1min, mobile phase A volume ratio is 70%, mobile phase B volume ratio is 30%;

[0068] 30min, the volume ratio of mobile phase A is 70%, and the volume ratio of mobile phase B is 30%.

[0069] Accurately measure the reference solution and sample solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the content of the nitrosamine impurity N-nitrosobumetanide in bumetanide using the impurity external standard method;

[0070] (4) Calculation method

[0071] The impurity content is calculated according to the following formula:

[0072]

[0073] A i = peak area of ​​impurities in sample solution;

[0074] A STD =5 for the average peak area of ​​N-nitrosobumetanide in the reference solution;

[0075] m STD = the weight of the impurity reference substance, mg;

[0076] mSpl = sample weight, mg;

[0077] V1 = dilution volume of impurity reference substance, ml;

[0078] V2 = dilution volume of sample, ml;

[0079] P = reference substance content;

[0080] The bumetanide is 3-butylamino-4-phenoxy-5-sulfamoylbenzoic acid, and its chemical structure is as follows:

[0081]

[0082] The chemical structural formula of the N-nitroso bumetanide is shown in Formula I:

[0083]

[0084] Effect Example

[0085] 1. Determination of the Chemical Structure of the Nitrosamine Impurity N-Nitroso-Bumetanide in Bumetanide of the Present Invention

[0086] The nitrosamine impurities in bumetanide of the present invention 1 H-NMR spectrum, 13 C-NMR spectrum and mass spectrum are shown in Figure 1 to Figure 3 As shown, it can be confirmed that the nitrosamine impurity is N-nitrosobumetanide.

[0087] II. Methodological Validation of the Method for Determining the Nitrosamine Impurity N-Nitroso-Bumetanide in Bumetanide of the Present Invention

[0088] 1. Specificity - sample matrix and known impurities

[0089] N-nitroso bumetanide stock solution: accurately weigh 3 mg of N-nitroso bumetanide, place in a 20 ml volumetric flask, dissolve and dilute to the mark with NMP, and shake well; accurately measure 2 ml and place in a 100 ml volumetric flask, dilute to the mark with NMP, and shake well to prepare a solution containing 3.0 μg of N-nitroso bumetanide per 1 ml.

[0090] Spiked sample solution: Take 80 mg of bumetanide sample, accurately weigh it, place it in a 20 ml volumetric flask, add 10 ml of NMP to dissolve it, accurately add 2 ml of BU304 stock solution, add appropriate amount of water, quickly cool it to room temperature in an ice water bath, dilute it to the scale with water, and shake well.

[0091] Take the prepared diluent, reference solution, sample solution, and spiked sample solution and inject them into the liquid chromatograph respectively. Record the chromatogram. The key spectra are shown in Figures 4 to 7 .in Figure 4 is the chromatogram of the diluent, Figure 5 is the chromatogram of the reference solution. Figure 6 is the chromatogram of the sample solution, Figure 7 The chromatogram is the spiked sample solution.

[0092] The results showed that the diluent had no interference; in the spiked sample solution, the separation between the N-nitroso bumetanide peak and the adjacent impurity peak was not less than 1.5 (2.36), and the purity angle of the N-nitroso bumetanide peak was less than the purity threshold. The peak elution time was moderate, the peak shape was symmetrical, and the separation effect was good, meeting the requirements.

[0093] 2. Detection limit and quantification limit test

[0094] The signal-to-noise ratio method was used to determine the detection limit and quantification limit. Prepare the N-nitroso bumetanide reference stock solution, dilute it to a certain concentration, inject it, and record the chromatogram. The sample detection amount when the signal-to-noise ratio S / N ≥ 10 is the quantification limit, and the sample detection amount when the signal-to-noise ratio S / N ≥ 3 is the detection limit. The chromatogram of the quantification limit solution is shown in Figure 8 , the chromatogram of the detection limit solution is shown in Fig. 9 , the specific results are shown in Table 1.

[0095] Table 1 Detection limit and quantification limit test results of the present invention

[0096]

[0097] The results showed that this method was sensitive to N-nitrosobumetanide and could accurately detect the content of N-nitrosobumetanide in bumetanide.

[0098] 3. Linearity test

[0099] Take the N-nitroso bumetanide reference substance and dilute it with diluent to make a linear stock solution containing 3 μg N-nitroso bumetanide per 1 ml.

[0100] A total of 7 portions of linear solutions were prepared with diluent to prepare LOQ, 50%, 80%, 100%, 120%, 150%, and 200% linear solutions equivalent to the limit concentration (75 ppm). The samples were injected and the chromatograms were recorded. The standard curve was drawn with the concentration as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 2.

[0101] Table 2 Linear test results of the present invention

[0102] Linear range (μg / ml) Linear equations Correlation coefficient (r) N-Nitrosobumetanide 0.0294～0.5877 y=82038.9284x+195.8543 0.9999

[0103] The linear curve equation of N-nitrosobumetanide is y=82038.9284x+195.8543, and the correlation regression coefficient r=0.9999.

[0104] The results showed that the method showed good linearity in the concentration range of 0.0294-0.5877 μg / ml for N-nitroso bumetanide.

[0105] 4. Accuracy test

[0106] The N-nitroso bumetanide reference substance was added to the bumetanide sample for sample recovery determination. The bumetanide sample solution (unspiked, concentration 4 mg / ml) was prepared for determination, and three copies were prepared in parallel to obtain the content of N-nitroso bumetanide in the sample. The limit of N-nitroso bumetanide was set at 75 ppm, and the research concentration was 50%, 100%, and 150% of the relative limit. Three copies were prepared in parallel for each concentration and the measured value was obtained. The recovery was calculated by dividing the difference between the measured value and the content in the sample by the amount of reference substance added. The results are shown in Table 3.

[0107] Table 3 Accuracy test results of the present invention

[0108] concentration 50% concentration 100% concentration 150% concentration Average recovery rate 101.9％ 100.4％ 101.0％ RSD 0.62％ 0.21％ 0.30％

[0109] The results showed that the recovery rates of individual samples were between 100.2% and 102.6%, indicating that the method had good accuracy.

[0110] 5. Precision test

[0111] (1) Repeatability: The precision of the results was determined by the same analyst under the same conditions. Six bumetanide spiked solutions were prepared in parallel and measured under conditions as similar as possible. The measured amount and RSD were calculated.

[0112] (2) Intermediate precision: To examine the effect of random variation on precision, another analyst prepared six bumetanide spiked solutions in parallel on different dates, calculated the measured amount and RSD, and combined the results with the 12 determinations from the repeatability test.

[0113] The results showed that the repeatability of 6 samples was 76.3ppm, and the RSD was 0.59%. The intermediate precision of 6 samples was 79.1ppm, and the RSD was 1.2%. The repeatability and intermediate precision results were combined, and the average N-nitroso bumetanide content of 12 samples was 77.7ppm, and the RSD was 2.1%. This shows that the precision of this method is good.

[0114] 6. Durability test

[0115] (1) Solution stability: Take the spiked sample solution and inject it for testing at 0, 7, 11, 17, 22, and 27 hours, record the chromatogram, and calculate the content ratio of N-nitrosobumetanide to that at 0 hour.

[0116] The results showed that when the spiked sample solution was placed in a sealed condition at room temperature, the ratio of N-nitroso bumetanide at each time point within 27 hours to the content at 0 hours was 101.7% to 105.1%. The results showed that when the spiked sample solution was placed in a sealed condition at room temperature, it was stable within 27 hours.

[0117] (2) Durability study: By deliberately changing the method parameters, the ability of the method to be unaffected by the changes in these parameters was examined. In this study, the following parameters were independently examined: column temperature (35±5℃), column flow rate (1.0±0.1ml / min), and the difference between the measured amount of the determined component and the measured amount under the initial conditions was examined. The spiked sample solution (two parallel copies) was injected into the liquid chromatograph and the chromatogram was recorded. The results are shown in Table 4.

[0118] Table 4 Durability study test results of the present invention

[0119] N-Nitrosobumetanide (ppm) RAD(%) compared with normal condition Normal conditions 76.3 / Column temperature (30℃) 85.4 5.6 Column temperature (40℃) 83.9 4.7 Flow rate (0.9ml / min) 84.6 5.2 Flow rate (1.1ml / min) 87.4 6.8

[0120] The results showed that after changing the column temperature (±5°C) and flow rate (±0.1ml / min), the relative average deviation of the content of N-nitrosobumetanide under each condition compared with the normal condition met the recognition standard.

[0121] From the above results, it can be seen that the method of the present invention can be used to effectively detect the nitrosamine impurity N-nitrosobumetanide in bumetanide. The method has strong specificity, good repeatability, good accuracy, and high sensitivity, can ensure the controllability of N-nitrosobumetanide in bumetanide, and can be applied to the quality control of bumetanide.

[0122] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for determining nitrosamine impurity N-nitrosobumetanide in bumetanide, characterized in that: The steps include: (1) Preparation of reference solution: Take the nitrosamine impurity N-nitrosobumetanide reference substance and add diluent to prepare a reference solution containing 0.3 μg N-nitrosobumetanide per 1 ml; (2) Preparation of sample solution: Take bumetanide API and add diluent to prepare a sample solution with a concentration of 4 mg / ml; (3) Chromatographic conditions: A high performance liquid chromatograph was used with an ultraviolet detector. The chromatographic column packing was octadecylsilane bonded silica gel. Mobile phase A was formic acid solution, mobile phase B was methanol, and gradient elution was performed. The gradient elution parameters were set as follows: From 0 to 20 min, the volume proportion of mobile phase A changed from 70% to 20%, and the volume proportion of mobile phase B changed from 30% to 80%; 20-25 min, mobile phase A volume ratio is 20%, mobile phase B volume ratio is 80%; From 25 to 25.1 min, the volume proportion of mobile phase A changed from 20% to 70%, and the volume proportion of mobile phase B changed from 80% to 30%; 25.1~30min, the volume proportion of mobile phase A is 70%, and the volume proportion of mobile phase B is 30%; Accurately measure the reference solution and sample solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the content of the nitrosamine impurity N-nitrosobumetanide in the bumetanide sample using the impurity external standard method; (4) Calculation method The impurity content is calculated according to the following formula: A i = peak area of ​​impurities in sample solution; A STD =5 for the average peak area of ​​N-nitrosobumetanide in the reference solution; m STD = the weight of the impurity reference substance, mg; m Spl = sample weight, mg; V1 = dilution volume of impurity reference substance, ml; V2 = dilution volume of sample, ml; P = reference substance content.

2. The method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide according to claim 1, characterized in that: In step (1) and step (2), the ratio of the diluent is NMP: water = 60:

40.

3. The method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide according to claim 2, characterized in that: Dilution step: First add 60% volume of NMP to fully dissolve, add appropriate amount of water to nearly the scale line, place in an ice water bath and quickly cool to room temperature, then make up to volume with water and shake well.

4. The method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide according to claim 1, characterized in that: In step (3), the detection wavelength of the ultraviolet detector is 258-262 nm.

5. The method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide according to claim 4, characterized in that: The detection wavelength of the ultraviolet detector is 260nm.

6. The method for determining the nitrosamine impurity N-nitroso bumetanide in bumetanide according to claim 1, characterized in that: In step (3), the column temperature of the chromatographic column is 30-40°C.

7. The method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide according to claim 6, characterized in that: The column temperature of the chromatographic column is 35°C.

8. The method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide according to claim 1, characterized in that: In step (3), the flow rate of the gradient elution is 0.9-1.1 ml / min.

9. The method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide according to claim 8, characterized in that: The flow rate of the gradient elution was 1.0 ml / min.

10. The method for determining the nitrosamine impurity N-nitrosobumetanide in bumetanide according to claim 1, characterized in that: In step (3), the gradient elution parameters are set as: 0min, the volume proportion of mobile phase A is 70%, and the volume proportion of mobile phase B is 30%; 20 min, mobile phase A volume ratio is 20%, mobile phase B volume ratio is 80%; 25 min, mobile phase A volume ratio is 20%, mobile phase B volume ratio is 80%; 25.1min, mobile phase A volume ratio is 70%, mobile phase B volume ratio is 30%; 30min, the volume ratio of mobile phase A is 70%, and the volume ratio of mobile phase B is 30%.

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