CN111965267A - Method for detecting genotoxic impurity aryl sulfonate in amlodipine besylate - Google Patents
Method for detecting genotoxic impurity aryl sulfonate in amlodipine besylate Download PDFInfo
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Abstract
The invention discloses a method for detecting genotoxic impurity aryl sulfonate in amlodipine besylate. The invention utilizes a derivatization headspace gas chromatography to analyze and detect aryl sulfonate in amlodipine besylate, adopts a gas chromatography peak area external standard method to determine the content of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate by measuring methyl iodide, ethyl benzenesulfonate and iodomethane, iodoethane and iodoisopropane generated by derivatization reaction of sodium iodide and methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, increases the quality controllability of medicaments, is a method for determining aryl sulfonate with good separation degree, strong specificity and high sensitivity, and provides a more direct and stable analysis method for detecting genotoxic impurities in amlodipine besylate.
Description
Technical Field
The invention belongs to the technical field of medicine quality detection, and particularly relates to a method for detecting genotoxic impurity aryl sulfonate in amlodipine besylate.
Background
The chemical name of amlodipine besylate is 3-ethyl-5-methyl-2- (2-aminoethoxymethyl) -4- (2-chlorphenyl) -1, 4-dihydro-6-methyl-3, 5-pyridine dicarboxylate benzene sulfonate, which is a third-generation long-acting dihydro pyridine calcium antagonist and a common clinical medicine for treating cardiovascular diseases such as angina and hypertension. Compared with other similar medicines, the medicine has the characteristics of high bioavailability, good oral absorption, no food influence, low dosage, long medicine effect maintenance time and the like. At present, amlodipine besylate is used as a first-line medicine for clinical treatment of angina and hypertension. Aryl sulfonate Methyl Benzenesulfonate (MBS), Ethyl Benzenesulfonate (EBS) and Isopropyl Benzenesulfonate (IBS) are genotoxic impurities possibly existing in amlodipine benzenesulfonate.
In recent years, genotoxic impurities are the focus of attention, and sulfonic acid substances such as methanesulfonic acid, methanesulfonic acid and the like and trace lower alcohol generate aryl sulfonate such as Methyl Benzenesulfonate (MBS), Ethyl Benzenesulfonate (EBS) and Isopropyl Benzenesulfonate (IBS) in a synthesis reaction, and alkyl sulfonates such as Methyl Methanesulfonate (MMS), Ethyl Methanesulfonate (EMS), Isopropyl Methanesulfonate (IMS), n-butyl methanesulfonate (NBMS), these substances can be alkylated with DNA and thus may be a cause of cancer, so that they are potentially genotoxic impurities, and it is important to control the threshold of toxicological interest (TTC) level of such impurities, which are strictly regulated by European Medicine Evaluation Agency (EMEA), the U.S. Food and Drug Administration (FDA) and international conference on drug registration (ICH).
Because the detection method and limit of aryl sulfonate in amlodipine besylate raw materials and preparations have no national standard, the residual quantity of Methyl Benzenesulfonate (MBS), Ethyl Benzenesulfonate (EBS) and Isopropyl Benzenesulfonate (IBS) in samples is necessary to be detected, and the method adopts sodium iodide derivatization reaction to determine the content of Methyl Benzenesulfonate (MBS), Ethyl Benzenesulfonate (EBS) and Isopropyl Benzenesulfonate (IBS) by detecting methyl iodide, ethyl iodide and 2-iodopropane.
Disclosure of Invention
The invention aims to provide a method for detecting genotoxic impurities of aryl sulfonate in amlodipine besylate, which has the advantages of strong specificity to each impurity of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, high sensitivity, good method precision, stable solution within 24 hours, sensitive and rapid detection, and accurate and reliable result, thereby realizing the determination of aryl sulfonate in amlodipine besylate and the quality control of raw materials and preparations thereof.
The method for detecting the genotoxic impurity aryl sulfonate in the amlodipine besylate comprises the following steps: analyzing and detecting aryl sulfonate in amlodipine besylate by using a derivatization headspace gas chromatography, which comprises the following specific steps:
(1) preparing a test solution: dissolving the sample with acetonitrile, performing ultrasonic treatment for 5-10min, fixing the volume, adjusting the concentration of amlodipine besylate to 5-20mg/ml, preferably 10mg/ml, and filtering; precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a sample solution;
(2) preparing a reference substance solution: preparing a methyl benzenesulfonate reference stock solution, an ethyl benzenesulfonate reference stock solution and an isopropyl benzenesulfonate reference stock solution with the concentration of 0.5-2ug/ml, preferably 1ug/ml, respectively by using acetonitrile; precisely measuring 2ml of each contrast medium stock solution respectively, placing in a 20ml headspace bottle, precisely adding 3ml of water and 6g of sodium iodide, sealing, and shaking up to obtain a contrast medium solution;
(3) measurement: respectively taking a test solution and a reference solution, carrying out headspace sample injection according to a method, collecting a chromatogram, and calculating a result by using a peak area external standard method; calculating the formula: ci ═ Cs ═ Ai/As; wherein Ai is the peak area of each impurity in the test sample; as is the peak area of the impurity reference substance; cs is the concentration of the impurity reference substance; ci is the concentration of the test sample.
The derivatization headspace gas chromatography adopts instruments and equipment such as an Agilent7890B gas chromatograph and an Agilent7697A headspace sample injector.
The chromatographic conditions for the derivatized headspace gas chromatography were as follows:
a chromatographic column: a chromatographic column taking polyethylene glycol PEG-20M cross-linked polymer as a stationary phase;
column temperature: an initial temperature of 3555 ℃, preferably 40 ℃, for 10 minutes, and a temperature of 160 ℃ at a rate of 10-25 ℃, preferably 20 ℃ per minute, for 3 minutes;
column flow rate: 1.0-3.0ml/min, preferably 1.5 ml/min;
a detector: a hydrogen flame ionization detector, 240-;
a sample inlet: shunting or not at 90-120 deg.C, preferably 110 deg.C;
the split ratio is as follows: 20:1 to 1:1, preferably 10: 1;
and (3) sample introduction mode: carrying out headspace sample injection;
headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
The chromatographic column comprises: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5. mu.m.
The method has the beneficial effects that: the method adopts a gas chromatography peak area external standard method to determine the content of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate by measuring methyl iodide, ethyl benzenesulfonate and isopropyl iodopropane generated by derivatization reaction of sodium iodide and methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, increases the controllability of the quality of the medicine, is a method for measuring aryl sulfonate with good separation degree, strong specificity and high sensitivity, and provides a more direct and stable analysis method for detecting genotoxic impurities in amlodipine benzenesulfonate.
Drawings
FIG. 1 GC diagram of the solvent of example 1 of the present invention.
FIG. 2 GC graph of the amlodipine besylate test sample solution of example 1 of the present invention.
FIG. 3 is a GC chart of an arylsulfonate-based control of example 1 of the invention.
FIG. 4 is a GC graph of an arylsulfonate-based control of example 2 of the invention.
FIG. 5 is a GC graph of an arylsulfonate-based control of example 3 of the invention.
FIG. 6 is a GC graph of an arylsulfonate-based control of example 4 of the invention.
FIG. 7 is a GC graph of an arylsulfonate-based control of example 5 of the invention.
FIG. 8 is a GC graph of an arylsulfonate-based control of example 6 of the invention.
FIG. 9 GC graph of an arylsulfonate-based control of example 7 of the invention.
FIG. 10 GC graph of the solvent of example 8 of the present invention.
FIG. 11 is a GC chart of the amlodipine besylate test sample solution of example 8 of the present invention.
FIG. 12 is a GC chart of a mixed solution of a control of example 8 of the present invention.
FIG. 13 GC graph of a control solution of methyl benzenesulfonate of example 8 of the present invention.
FIG. 14 GC graph of a control solution of ethyl benzenesulfonate of example 8 of the present invention.
FIG. 15 GC graph of a control solution of isopropyl benzenesulfonate of example 8 of the present invention.
FIG. 16 GC graph of a control solution for precision testing of example 8 of the present invention.
FIG. 17 GC graph of a control solution for precision testing of example 8 of the present invention.
FIG. 18 GC graph of a control solution for precision testing of example 8 of the present invention.
FIG. 19 is a GC graph of a control solution for precision testing of example 8 of the present invention.
FIG. 20 GC graph of a control solution for precision testing of example 8 of the present invention.
FIG. 21 GC graph of a control solution for the linear assay of example 8 of the present invention.
FIG. 22 GC graph of a control solution of the linear test of example 8 of the present invention.
FIG. 23 GC graph of a control solution for the linear assay of example 8 of the present invention.
FIG. 24 GC graph of a control solution for the linear assay of example 8 of the present invention.
FIG. 25 GC graph of a control solution of the linear assay of example 8 of the present invention.
FIG. 26 GC graph of a control solution of the linear test of example 8 of the present invention.
FIG. 27 GC graph of a detection limit test control solution in example 8 of the present invention.
FIG. 28 GC graph of a control solution for the quantitative limit test of example 8 of the present invention.
FIG. 29 GC graph of the spiked (low concentration) test sample solution for the recovery test of example 8 of the present invention.
FIG. 30 GC plots of the spiked (low concentration) test sample solutions for the recovery test of example 8 of the present invention.
FIG. 31 GC graph of the spiked (low concentration) test sample solution for the recovery test of example 8 of the present invention.
FIG. 32 GC plots of the spiked (medium concentration) test sample solutions for the recovery test of example 8 of the present invention.
FIG. 33 GC plots of the spiked (medium concentration) test sample solutions for the recovery test of example 8 of the present invention.
FIG. 34 GC graph of a spiked (medium concentration) test sample solution for the recovery test of example 8 of the present invention.
FIG. 35 GC graph of a sample solution of the recovery test spiked (high concentration) of example 8 of the present invention.
FIG. 36 GC graph of the spiked (high concentration) test sample solution for the recovery test of example 8 of the present invention.
FIG. 37 GC plots of the spiked (high concentration) test sample solutions for the recovery test of example 8 of the present invention.
FIG. 38 GC graph of a control solution after being left for 0 hours for stability testing in example 8 of the present invention.
FIG. 39 GC graph of a control solution of example 8 of the present invention placed in a stability test for 2 hours.
FIG. 40 GC graph of a control solution of example 8 of the present invention placed in a stability test for 4 hours.
FIG. 41 GC graph of a control solution after being left for 18 hours for stability testing in example 8 of the present invention.
FIG. 42 GC graph of a control solution of example 8 of the present invention placed in a stability test for 20 hours.
FIG. 43 GC graph of a control solution of example 8 of the present invention placed in a stability test for 24 hours.
FIG. 44 is a linear relationship diagram of the linear test of the genotoxic impurity MBS in example 8 of the present invention.
FIG. 45 is a linear relationship chart of the linear test of the genotoxic impurity EBS in example 8 of the present invention.
FIG. 46 is a linear relationship chart of IBS linear assay for genotoxic impurity in example 8 of the present invention.
Detailed Description
Example 1:
(1) preparing a test solution: taking a proper amount of samples, precisely weighing, dissolving with a proper amount of acetonitrile, carrying out ultrasonic treatment for 10min, fixing the volume, then measuring 10mg/ml according to the amlodipine besylate, and filtering. Precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a sample solution;
(2) preparing a reference substance solution: taking a proper amount of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, precisely weighing, and dissolving and diluting with acetonitrile to obtain a reference stock solution with methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate concentrations of 1 ug/ml. Precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a reference solution;
sampling the above solvent, sample solution, and reference solution respectively in headspace, collecting chromatogram (shown in fig. 1, 2, and 3), and calculating the result by peak area external standard method.
The chromatographic conditions were as follows:
a chromatographic column: the polyethylene glycol (PEG-20M) cross-linked polymer is taken as a stationary phase, and the following is recommended: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5 μm;
column temperature: the initial temperature was 40 ℃, maintained for 10 minutes, and the temperature was raised to 160 ℃ at a rate of 20 ℃ per minute, maintained for 3 minutes;
column flow rate: 2.0 ml/min;
sample inlet temperature: split/no split, temperature 110 ℃;
a detector: hydrogen Flame Ionization Detector (FID), 250 ℃;
the split ratio is as follows: 10: 1
And (3) sample introduction mode: headspace sampling
Headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
Wherein FIG. 1 is a solvent peak of a blank solvent acetonitrile; FIG. 2 is a chromatogram peak of a test sample solution; fig. 3 is a reference solution, wherein the retention times of methyl iodide, ethyl iodide and isopropyl iodide obtained by derivatization reaction of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate with methyl iodide are respectively: and 2.967, 3.915 and 4.419min, determining the existence of the methyl benzenesulfonate, the ethyl benzenesulfonate and the isopropyl benzenesulfonate according to the peak emergence time of the reference solution and the test solution, and determining the contents of the methyl benzenesulfonate, the ethyl benzenesulfonate and the isopropyl benzenesulfonate in the test solution according to the peak emergence area.
Example 2:
preparing a reference substance solution: taking a proper amount of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, precisely weighing, and dissolving and diluting with acetonitrile to obtain a reference stock solution with methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate concentrations of 1 ug/ml. Precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a reference solution;
sampling the above reference solutions via headspace, collecting chromatogram (the reference solution is shown in FIG. 4), and calculating the result by peak area external standard method.
The chromatographic conditions were as follows:
a chromatographic column: the polyethylene glycol (PEG-20M) cross-linked polymer is taken as a stationary phase, and the following is recommended: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5 μm;
column temperature: the initial temperature was 40 ℃, maintained for 10 minutes, and the temperature was raised to 160 ℃ at a rate of 20 ℃ per minute, maintained for 3 minutes;
column flow rate: 2.0 ml/min;
sample inlet temperature: split/no split, temperature 105 ℃;
a detector: hydrogen Flame Ionization Detector (FID), 250 ℃;
the split ratio is as follows: 10: 1
And (3) sample introduction mode: headspace sampling
Headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
Example 3:
preparing a reference substance solution: taking a proper amount of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, precisely weighing, and dissolving and diluting with acetonitrile to obtain a reference stock solution with methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate concentrations of 1 ug/ml. Precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a reference solution;
sampling the above reference solutions via headspace, collecting chromatogram (the reference solution is shown in FIG. 5), and calculating the result by peak area external standard method.
The chromatographic conditions were as follows:
a chromatographic column: the polyethylene glycol (PEG-20M) cross-linked polymer is taken as a stationary phase, and the following is recommended: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5 μm;
column temperature: the initial temperature was 40 ℃, maintained for 10 minutes, and the temperature was raised to 160 ℃ at a rate of 20 ℃ per minute, maintained for 3 minutes;
column flow rate: 2.0 ml/min;
sample inlet temperature: split/no split, temperature 115 ℃;
a detector: hydrogen Flame Ionization Detector (FID), 250 ℃;
the split ratio is as follows: 10: 1
And (3) sample introduction mode: headspace sampling
Headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
Example 4:
preparing a reference substance solution: taking a proper amount of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, precisely weighing, and dissolving and diluting with acetonitrile to obtain a reference stock solution with methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate concentrations of 1 ug/ml. Precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a reference solution;
sampling the above reference solutions via headspace, collecting chromatogram (the reference solution is shown in FIG. 6), and calculating the result by peak area external standard method.
The chromatographic conditions were as follows:
a chromatographic column: the polyethylene glycol (PEG-20M) cross-linked polymer is taken as a stationary phase, and the following is recommended: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5 μm;
column temperature: the initial temperature was 40 ℃, maintained for 10 minutes, and the temperature was raised to 160 ℃ at a rate of 20 ℃ per minute, maintained for 3 minutes;
column flow rate: 1.95 ml/min;
sample inlet temperature: split/no split, temperature 110 ℃;
a detector: hydrogen Flame Ionization Detector (FID), 250 ℃;
the split ratio is as follows: 10: 1
And (3) sample introduction mode: headspace sampling
Headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
Example 5:
preparing a reference substance solution: taking a proper amount of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, precisely weighing, and dissolving and diluting with acetonitrile to obtain a reference stock solution with methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate concentrations of 1 ug/ml. Precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a reference solution;
sampling the above reference solutions via headspace, collecting chromatogram (the reference solution is shown in FIG. 7), and calculating the result by peak area external standard method.
The chromatographic conditions were as follows:
a chromatographic column: the polyethylene glycol (PEG-20M) cross-linked polymer is taken as a stationary phase, and the following is recommended: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5 μm;
column temperature: the initial temperature was 40 ℃, maintained for 10 minutes, and the temperature was raised to 160 ℃ at a rate of 20 ℃ per minute, maintained for 3 minutes;
column flow rate: 2.05 ml/min;
sample inlet temperature: split/no split, temperature 110 ℃;
a detector: hydrogen Flame Ionization Detector (FID), 250 ℃;
the split ratio is as follows: 10: 1
And (3) sample introduction mode: headspace sampling
Headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
Example 6:
preparing a reference substance solution: taking a proper amount of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, precisely weighing, and dissolving and diluting with acetonitrile to obtain a reference stock solution with methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate concentrations of 1 ug/ml. Precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a reference solution;
sampling the above reference solutions via headspace, collecting chromatogram (the reference solution is shown in FIG. 8), and calculating the result by peak area external standard method.
The chromatographic conditions were as follows:
a chromatographic column: the polyethylene glycol (PEG-20M) cross-linked polymer is taken as a stationary phase, and the following is recommended: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5 μm;
column temperature: the initial temperature was 40 ℃, maintained for 10 minutes, and the temperature was raised to 160 ℃ at a rate of 20 ℃ per minute, maintained for 3 minutes;
column flow rate: 2.0 ml/min;
sample inlet temperature: split/no split, temperature 110 ℃;
a detector: hydrogen Flame Ionization Detector (FID), 245 ℃;
the split ratio is as follows: 10: 1
And (3) sample introduction mode: headspace sampling
Headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
Example 7:
preparing a reference substance solution: taking a proper amount of methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, precisely weighing, and dissolving and diluting with acetonitrile to obtain a reference stock solution with methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate concentrations of 1 ug/ml. Precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a reference solution;
sampling the above reference solutions via headspace, collecting chromatogram (the reference solution is shown in FIG. 9), and calculating the result by peak area external standard method.
The chromatographic conditions were as follows:
a chromatographic column: the polyethylene glycol (PEG-20M) cross-linked polymer is taken as a stationary phase, and the following is recommended: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5 μm;
column temperature: the initial temperature was 40 ℃, maintained for 10 minutes, and the temperature was raised to 160 ℃ at a rate of 20 ℃ per minute, maintained for 3 minutes;
column flow rate: 2.0 ml/min;
sample inlet temperature: split/no split, temperature 110 ℃;
a detector: hydrogen Flame Ionization Detector (FID), 255 ℃;
the split ratio is as follows: 10: 1
And (3) sample introduction mode: headspace sampling
Headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
Example 8: the methodological verification test takes the example 1 as an example, and methodological verification is respectively carried out on the aspects of specificity, precision, linearity, detection limit, quantification limit, sample adding recovery rate and stability, and the results are as follows:
(1) specificity
Respectively taking acetonitrile, a test solution (amlodipine besylate), a reference mixed solution (MBS, EBS and IBS), an MBS reference solution, an EBS reference solution and an IBS reference solution, and sequentially injecting samples. The results show (fig. 10-15) that the solvent and the reference substance mixed solution (MBS, EBS and IBS) are well separated, the amlodipine besylate does not interfere with each impurity reference substance, and the method has strong specificity.
(2) Precision degree
Sampling the reference substance mixed solution (MBS, EBS, IBS) for 6 times according to the method, and calculating the precision (shown in figure 16-20):
as obtained in the table: the sample introduction of the reference substance mixed solution is carried out for 6 times, the relative deviation of the peak areas of MBS, EBS and IBS is respectively 7.6%, 8.6% and 6.1%, and the chromatographic condition has good precision because the relative deviation meets the calculation requirement of an external standard method.
(3) Linearity
Taking methyl benzenesulfonate, ethyl benzenesulfonate and isopropyl benzenesulfonate, weighing each 50mg precisely, placing in a 100ml measuring flask, dissolving and diluting to scale with acetonitrile, and shaking up. Precisely measuring 5ml, placing in a 100ml measuring flask, adding acetonitrile to dilute to scale, and shaking up to obtain a linear control stock solution; diluting the stock solution of linear control with acetonitrile to obtain 0.25ug/ml, 0.5ug/ml, 1.0ug/ml, 1.25ug/ml, and 1.5ug/ml concentration series solutions, precisely measuring 2ml of each solution, placing in a 20ml headspace bottle, precisely adding 3ml of water and 6g of sodium iodide, sealing, shaking to obtain a series of control solutions, introducing via headspace, and measuring the linear relationship of concentration (shown in figures 21-26)
And (4) conclusion: from FIGS. 44 to 46, it is clear that the genotoxic impurities MBS, EBS and IBS have good linearity in the concentration range of 0.25ug/ml to 1.5ug/ml, and R values are 0.9937, 0.9892 and 0.9898, respectively.
(4) Detection limit and quantitative limit (FIGS. 27-28)
Taking the reference substance mixed stock solution to gradually dilute the serial concentration solutions by acetonitrile, precisely measuring 2ml of each solution, placing the solution in a 20ml headspace bottle, precisely adding 3ml of water and 6g of sodium iodide, sealing, shaking uniformly to serve as the serial reference substance solution, carrying out headspace sample injection according to a method, and measuring the detection limit quantity of each genotoxic impurity as follows:
| name of impurity | Detection limit (ug/ml) | Quantitative limit (ug/ml) |
| Benzenesulfonic acid methyl ester | 0.10 | 0.26 |
| Benzenesulfonic acid ethyl ester | 0.10 | 0.24 |
| Benzene sulfonic acid isopropyl ester | 0.10 | 0.24 |
(5) Sample recovery rate
Adding the control mixed stock solution into the sample to obtain low, medium and high standard concentration solutions, and measuring the result by the method (shown in figure 29-37)
The results show that the sample adding recovery rates of the genotoxic impurities MBS, EBS and IBS measured under three concentrations are respectively 88-106%, 87-107% and 84-105%, and meet the calculation requirement of the recovery rate of the gas phase headspace method (80-120%),
the method is proved to be accurate and controllable.
(6) Stability of solution
Standing the control mixed solution for 24 hr, sampling samples at different time intervals, and obtaining the following results (figures 38-43)
From the results, the relative standard deviations of the solutions of the control samples of the genotoxic impurities MBS, EBS and IBS were 5.2%, 8.2% and 4.1%, respectively, and the solution was stable for 24 hours.
Claims (4)
1. A method for detecting genotoxic impurity aryl sulfonate in amlodipine besylate is characterized in that aryl sulfonate in amlodipine besylate is analyzed and detected by using a derivatization headspace gas chromatography, and the method comprises the following specific steps:
(1) preparing a test solution: dissolving the sample with acetonitrile, performing ultrasonic treatment for 5-10min, fixing the volume, adjusting the concentration of amlodipine besylate to 5-20mg/ml, preferably 10mg/ml, and filtering; precisely measuring 2ml, placing in a 20ml headspace bottle, precisely adding 3ml water and 6g sodium iodide, sealing, shaking to obtain a sample solution;
(2) preparing a reference substance solution: preparing a methyl benzenesulfonate reference stock solution, an ethyl benzenesulfonate reference stock solution and an isopropyl benzenesulfonate reference stock solution with the concentration of 0.5-2ug/ml, preferably 1ug/ml, respectively by using acetonitrile; precisely measuring 2ml of each contrast medium stock solution respectively, placing in a 20ml headspace bottle, precisely adding 3ml of water and 6g of sodium iodide, sealing, and shaking up to obtain a contrast medium solution;
(3) measurement: respectively taking a test solution and a reference solution, carrying out headspace sample injection according to a method, collecting a chromatogram, and calculating a result by using a peak area external standard method; calculating the formula: ci ═ Cs ═ Ai/As; wherein Ai is the peak area of each impurity in the test sample; as is the peak area of the impurity reference substance; cs is the concentration of the impurity reference substance; ci is the concentration of the test sample.
2. The method for detecting the genotoxic impurity aryl sulfonate in the amlodipine besylate according to claim 1, wherein the instruments used for the derivatization headspace gas chromatography are Agilent7890B gas chromatograph and Agilent7697A headspace sample injector.
3. The method for detecting the genotoxic impurity aryl sulfonate in the amlodipine besylate according to claim 1, wherein the chromatographic conditions of the derivatization headspace gas chromatography are as follows:
a chromatographic column: a chromatographic column taking polyethylene glycol PEG-20M cross-linked polymer as a stationary phase;
column temperature: an initial temperature of 3555 ℃, preferably 40 ℃, for 10 minutes, and a temperature of 160 ℃ at a rate of 10-25 ℃, preferably 20 ℃ per minute, for 3 minutes;
column flow rate: 1.0-3.0ml/min, preferably 1.5 ml/min;
a detector: a hydrogen flame ionization detector, 240-;
a sample inlet: shunting or not at 90-120 deg.C, preferably 110 deg.C;
the split ratio is as follows: 20:1 to 1:1, preferably 10: 1;
and (3) sample introduction mode: carrying out headspace sample injection;
headspace conditions: the equilibrium temperature of the headspace bottle is 80 ℃, the equilibrium time is 30 minutes, the quantitative loop temperature is 90 ℃, the transmission line temperature is 100 ℃, and the sample injection amount is 1 ml.
4. The method for detecting the genotoxic impurity aryl sulfonate in amlodipine besylate according to claim 3, wherein the chromatographic column is: agilent DB-Wax, 30 m.times.0.320 mm.times.0.5. mu.m.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114216985A (en) * | 2021-12-22 | 2022-03-22 | 珠海润都制药股份有限公司 | Method for testing diisopropyl sulfate in isosorbide |
| CN114460203A (en) * | 2022-02-15 | 2022-05-10 | 浙江海正药业股份有限公司 | Method for simultaneously detecting methyl p-toluenesulfinate, ethyl p-toluenesulfinate and isopropyl p-toluenesulfinate in medicine |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481348A (en) * | 2009-01-07 | 2009-07-15 | 南昌弘益科技有限公司 | Production method of levamlodipine besylate |
| CN107632078A (en) * | 2017-08-14 | 2018-01-26 | 中国科学院上海药物研究所 | A kind of method of methyl tosylate in measure medicine |
| CN111089931A (en) * | 2019-11-28 | 2020-05-01 | 上海秀新臣邦医药科技有限公司 | Detection method of parecoxib sodium gene genotoxicity impurity |
| CN111307958A (en) * | 2019-11-25 | 2020-06-19 | 澳美制药(苏州)有限公司 | Method for detecting genotoxic impurities in bepotastine besilate |
-
2020
- 2020-06-30 CN CN202010615273.2A patent/CN111965267A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481348A (en) * | 2009-01-07 | 2009-07-15 | 南昌弘益科技有限公司 | Production method of levamlodipine besylate |
| CN107632078A (en) * | 2017-08-14 | 2018-01-26 | 中国科学院上海药物研究所 | A kind of method of methyl tosylate in measure medicine |
| CN111307958A (en) * | 2019-11-25 | 2020-06-19 | 澳美制药(苏州)有限公司 | Method for detecting genotoxic impurities in bepotastine besilate |
| CN111089931A (en) * | 2019-11-28 | 2020-05-01 | 上海秀新臣邦医药科技有限公司 | Detection method of parecoxib sodium gene genotoxicity impurity |
Non-Patent Citations (3)
| Title |
|---|
| 侯奇伟 等: "衍生气相色谱法测定草酸艾司西酞普兰中甲磺酸烷基酯类", 《广东化工》 * |
| 李靖坤等: "气相色谱-质谱联用法测定对甲苯磺酸脂类基因毒性杂质的方法学验证 ", 《当代化工》 * |
| 连小刚 等: "衍生气相色谱法测定甲磺酸伊马替尼中甲磺酸烷基酯类", 《中国医药科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114216985A (en) * | 2021-12-22 | 2022-03-22 | 珠海润都制药股份有限公司 | Method for testing diisopropyl sulfate in isosorbide |
| CN114460203A (en) * | 2022-02-15 | 2022-05-10 | 浙江海正药业股份有限公司 | Method for simultaneously detecting methyl p-toluenesulfinate, ethyl p-toluenesulfinate and isopropyl p-toluenesulfinate in medicine |
| CN114460203B (en) * | 2022-02-15 | 2023-12-22 | 浙江海正药业股份有限公司 | Method for simultaneously detecting methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in medicine |
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