CN114088843A - Method for detecting nitrosamine genotoxic impurities in varenicline intermediate - Google Patents
Method for detecting nitrosamine genotoxic impurities in varenicline intermediate Download PDFInfo
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- CN114088843A CN114088843A CN202111418224.0A CN202111418224A CN114088843A CN 114088843 A CN114088843 A CN 114088843A CN 202111418224 A CN202111418224 A CN 202111418224A CN 114088843 A CN114088843 A CN 114088843A
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- 238000000034 method Methods 0.000 title claims abstract description 34
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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Abstract
The invention provides a method for detecting nitrosamine genotoxic impurities in varenicline intermediates, which comprises the steps of determining the content of nitrosamine genotoxic impurities by adopting a high-resolution liquid chromatography and mass spectrometry combined technology, wherein the genotoxic impurities of the genes are compounds A, B, C, and the chemical structural formulas of the compounds are respectively as follows:
the varenicline intermediate has the following structure:
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting nitrosamine genotoxic impurities in varenicline intermediates.
Background
Varenicline is a drug developed by the U.S. pfeir company for the treatment of nicotine addiction. At present, a great deal of reports are made about the preparation and synthesis of varenicline intermediates, but the attention on genotoxic impurities is less.
In original patent CN101410110A, varenicline may have one or more of several mononitro, monoamino, mixed aminonitro, diamino or dinitro intermediate impurities, but no research is made on nitroso group impurities.
In 6 months of 2021, due to unacceptable nitrosamine content, pfurre stopped the production of the smoking cessation drug Chantix (varenicline), N-nitroso-varenicline. Currently, there is increasing interest in genotoxicity testing in varenicline. Therefore, a detection method for genotoxic impurities in the drug synthesis process with high sensitivity and good specificity is developed, particularly, intermediate nitrosamine genotoxic impurities are strictly controlled, and the accurate and effective control on the varenicline drug quality can be realized, so that the drug safety of varenicline patients is ensured, and the method has great research significance.
The invention content is as follows:
the method aims to solve the problems of scarcity of a method for detecting genotoxic impurities of varenicline intermediates and safety of varenicline medication. The invention provides a method for detecting nitrosamine genotoxic impurities in varenicline intermediate, wherein the varenicline intermediate is shown in formula I, and the chemical name of the varenicline intermediate is 2,3,4, 5-tetrahydro-7-nitro-3- (trifluoroacetyl) -1, 5-methano-1-hydro-3-benzonitazone
The structure is as follows:
the invention provides a method for detecting nitrosamine genotoxic impurities in varenicline intermediates, which adopts a high-resolution liquid chromatography and mass spectrometry combined technology to determine nitrosamine genotoxic impurities.
Further, the method for detecting the genotoxic impurity of nitrosamine in varenicline intermediate is characterized in that the genotoxic impurity is compound A, B, C, and the chemical structural formulas of the genotoxic impurity are respectively as follows:
the invention provides a detection method for nitrosamine genotoxic impurities in varenicline intermediates, which adopts high-resolution liquid chromatography and mass spectrometry combined technology for determination and comprises the following detection methods:
(1) chromatographic conditions
The filler is an octadecyl bonded silica gel chromatographic column,
the mobile phase is 0.1 percent formic acid water solution and 0.1 percent formic acid acetonitrile solution with a certain volume ratio, gradient elution is carried out,
the column temperature is 40-50 ℃,
the flow rate of the mobile phase is 0.4-0.6 mL/min,
the detection wavelength is 210-230 nm.
(2) Conditions of Mass Spectrometry
The ion source is a heatable electrospray ionization source, and the temperature of the ion source is 400-500 DEG C
The sheath gas flow rate is 50 to 60arbitrary units, the auxiliary gas flow rate is 10 to 20arbitrary units,
the collision energy is 50 to 70(NCE)
The temperature of the ion transmission tube is 300-400 ℃,
the scan mode is a positive ion mode,
the collection mode is a parallel reaction monitoring mode or a selective ion monitoring mass spectrometry mode.
Further, the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate comprises the following sample preparation methods:
(3) sample preparation method
Preparing a reference substance solution: preparing 7.8ng of control solution containing the genotoxic impurity A, B, C in each 1 mL;
preparing a test solution: varenicline intermediate is precisely weighed and prepared into a solution containing varenicline with the concentration of 2mg/mL by using acetonitrile water solution (50:50, v/v).
Further, the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate comprises the following steps:
(4) detection method
Injecting the test solution and each reference solution into a high-resolution liquid chromatograph-mass spectrometer for detection.
Further, according to the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, the model of an octadecyl bonded silica gel chromatographic column in the chromatographic condition in the step (1) is Agilent _ ZORBAX Eclipse Plus C18-150 x4.6mm-3.5 mu m.
Further, in the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, in the chromatographic condition of the step (1), mobile phases of gradient elution are 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution with different volume ratios, and the following elution gradient is adopted:
| time (min) | 0.1% aqueous formic acid solution | 0.1% formic acid acetonitrile solution |
| 0.0 | 50％ | 50％ |
| 8.0 | 35％ | 65％ |
| 10.0 | 35％ | 65％ |
| 15.0 | 0％ | 100％ |
| 15.1 | 50％ | 50％ |
| 20.0-30.0 | 50％ | 50％ |
The above percentages are by volume.
Further, according to the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, the temperature of a chromatographic column is set to be 45 ℃ in the chromatographic condition of the step (1).
Further, according to the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, the flow rate of a mobile phase in the chromatographic condition in the step (1) is 0.5 mL/min.
Further, according to the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, the detection wavelength is 214nm in the chromatographic condition in the step (1).
Further, according to the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, in the mass spectrometry condition of the step (2), the temperature of an electrospray ionization source ion source is 420-480 ℃.
Further, according to the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, in the mass spectrum condition in the step (2), the flow rate of the sheath gas of the high-resolution mass spectrum is 55arbitrary units, and the auxiliary gas flow rate is 15arbitrary units.
Further, according to the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, in the mass spectrometry condition in the step (2), the temperature of an ion transmission tube is 360-390 ℃.
Further, in the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, in the step (3), in the sample preparation, a sample solution is prepared by taking the varenicline intermediate, and using water and acetonitrile in a volume ratio of 50:50 to prepare a solution containing 2mg/mL of varenicline.
Further, in the detection method of varenicline intermediate nitrosamine genotoxic impurities, in the step (4), a sample solution and each reference solution are injected into a high-resolution liquid chromatograph-mass spectrometer, about 1mL of the sample solution is filled in a 1.5mL sample injection small bottle, and a sample is collected and recorded by an automatic sample injection device of a high performance liquid chromatograph. By the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate, the nitrosamine genotoxic impurities in the varenicline intermediate shown as the formula I can be well detected, wherein the content of A (or the limit) can be controlled to be less than or equal to 3.9ppm, the content of B is controlled to be less than or equal to 3.9ppm, and the content of C is controlled to be less than or equal to 3.9 ppm; more preferably, the A content is 0.78ppm or less, the B content is 0.78ppm or less and the C content is 0.78ppm or less.
Compared with the prior art, the invention has better beneficial effects:
(1) the method for detecting the nitrosamine genotoxic impurities in the varenicline intermediate can effectively separate the main component peak and the genotoxic impurity peak of the varenicline intermediate, can provide higher specificity at the same time, and avoids detecting false positive impurities.
(2) The method detects genotoxic impurities in the varenicline intermediate through high-resolution liquid chromatography-mass spectrometry, and uses octadecylsilane chemically bonded silica as a filling agent.
(3) The detection method has the advantages that the mobile phase proportion has great influence on the separation degree of genotoxic impurities and varenicline in experimental results, and further research shows that the genotoxic impurities and varenicline intermediate can be accurately separated by setting mobile phases with different proportions to carry out gradient elution, and finally, the mobile phase proportion is that 0.1 percent of formic acid aqueous solution is 50 percent compared with 0.1 percent of formic acid acetonitrile solution in 0 to 8 minutes: 50-35: 65 in 8 to 10 minutes in a ratio of 35: 65 for 2 minutes, in 10 to 15 minutes, the 0.1% formic acid in acetonitrile rises from 65% to 100%, and the eluent ratio is then adjusted back to 50: 50.
(4) according to the detection method, the collision energy of the target ions has great influence on the sensitivity of genotoxic impurities in the detection result, and through research, the mass-to-charge ratio (189.0788) for quantification is found to have the maximum ion response when the collision energy of the compound A is 60(NCE), the mass-to-charge ratio (156.0808) for quantification is found to have the maximum ion response when the collision energy of the compound A is 50(NCE), and the mass-to-charge ratio (128.0628) for quantification is found to have the maximum ion response when the collision energy of the compound C is 70(NCE), so that the sample injection amount is 15 muL, the collision energy of the compound A is 60(NCE), the quantitative mass-to-charge ratio is 189.0788, the collision energy of the compound B is 50(NCE), the quantitative mass-to-charge ratio is 156.0808, the collision energy of the compound C is 70(NCE), and the quantitative mass-to-charge ratio is 128.0628.
(5) The detection method has high detection sensitivity, and the quantitative limit of A, B, C can reach 0.78ppm and is far lower than the index limit (3.9 ppm).
(6) The detection method can realize effective control of A, B, C genotoxic impurities in the varenicline intermediate, reduce the occurrence of side effects of medication of patients and ensure the medication safety of the patients to a certain extent.
Drawings
FIG. 1A, B, C overlay of liquid phase-high resolution mass spectrometry of three genotoxicities.
FIG. 2 blank spectrogram of liquid phase-high resolution mass spectrum of genotoxic impurity A
FIG. 3 liquid phase-high resolution mass spectrum of genotoxic impurity A
FIG. 4 blank spectrum of liquid phase-high resolution mass spectrum of genotoxic impurity B
FIG. 5 liquid phase-high resolution mass spectrum of genotoxic impurity B
FIG. 6 blank chromatogram of liquid phase-high resolution mass spectrum of genotoxic impurity C
FIG. 7 liquid phase-high resolution mass spectrum of genotoxic impurity C
Detailed Description
The acetonitrile in the mobile phase of the high performance liquid phase in the following examples is chromatographically pure acetonitrile. In the examples, the temperature is set temperature, the high efficiency liquid phase module allows the temperature error to be + -2 ℃, and the high resolution mass spectrum module allows the temperature error to be + -5 ℃, without specific description.
The detection method is an external standard method in the field of pharmaceutical analysis, and the following experimental processes and operation modes in the experimental method are not described in detail and all adopt standard operation procedures, such as solution preparation, operation methods of system applicability experiments and the like.
Example 1: method for groping
(1) Chromatographic conditions
The filler is an octadecyl bonded silica gel chromatographic column,
the mobile phase is 0.1 percent formic acid water solution and 0.1 percent formic acid acetonitrile solution with different volume ratios, and gradient elution is carried out according to the following table
The column temperature was 40 c,
the flow rate of the mobile phase is 0.4mL/min,
the detection wavelength was 214 nm.
(2) Conditions of Mass Spectrometry
The ion source is a heatable electrospray ionization source and has a temperature of 450 deg.C
The sheath gas flow rate is 55arbitrary units, the auxiliary gas flow rate is 15arbitrary units,
the collision energy is 50 to 70(NCE)
The temperature of the ion transmission tube is 380 ℃,
the scan mode is a positive ion mode,
the acquisition mode is a parallel reaction monitoring mode.
(3) Sample preparation
Preparing a reference substance solution: an appropriate amount of A, B, C genotoxic impurity control was precisely weighed, dissolved in acetonitrile aqueous solution (50:50, v/v), and prepared into 7.8ng control solutions per 1mL of the control solution containing genotoxic impurity A, B, C.
Preparing a test solution: varenicline intermediate is precisely weighed and prepared into a solution containing varenicline intermediate with the concentration of 2mg/mL by using acetonitrile water solution (50:50, v/v).
(4) Detection method
And injecting the test solution and each reference solution into a high-resolution liquid chromatograph-mass spectrometer.
The experimental results are as follows:
chromatogram results: the chromatogram contrast of A, B, C genotoxic impurity, the chromatogram of blank solution, and the chromatogram of each control solution obtained under the above chromatogram conditions are shown in FIGS. 1-7. The three genotoxic impurities obtained according to the chromatographic conditions described above can be effectively separated.
According to the analysis of the impurity reference substance in the varenicline intermediate, the impurity peak in the varenicline intermediate can be determined to be A, B, C genotoxic impurity.
Example 2 methodological investigation-System suitability
According to expert models and reasoning of software, the genotoxic impurity A, B, C is predicted to be 3 kinds of impurities in ICH, and the impurity research and control are required according to the guiding principle of ICH M7. Combining the Toxicological Threshold (TTC) data of 1.5. mu.g/day and the actual process conditions, it was finally confirmed that the limit of genotoxic impurity A, B, C was 3.9 ppm. According to the limit and the solubility of the varenicline intermediate, 1mL of control solution containing 7.8ng of genotoxic impurity A, B, C is prepared respectively, 6 needles are injected repeatedly when the system is balanced, mass spectrograms are recorded, and the system applicability test result is shown in table 1.
Example 3 methodological investigation-limits of quantitation
A limiting solution containing 7.8ng of genotoxic impurity A, 7.8ng of genotoxic impurity B and 7.8ng of genotoxic impurity C was prepared by diluting 5 times the control solution prepared in example 1, the signal-to-noise ratio of each impurity was greater than 10, and the results of the limiting assay are shown in Table 1.
Example 4 methodology examination-linearity and Range
Appropriate amounts of three genotoxic impurity reference substances are respectively weighed, the three genotoxic impurity reference substances are dissolved and diluted into appropriate concentrations by using a mixed solvent of water and acetonitrile, the solutions are taken for detection, and the results of linear and range tests are shown in table 1.
Example 5 methodological investigation-accuracy
Weighing a proper amount of varenicline intermediate, adding a proper amount of reference substance stock solution to obtain standard solution with the addition of each impurity of 50%, 100% and 150%, parallelly injecting 3 needles of solution at each concentration, recording mass spectrogram, and obtaining accuracy test results shown in table 1.
Example 6 methodological investigation-solution stability
Taking the reference substance solution under the system applicability item, placing at room temperature, injecting samples for testing at 0, 2, 4, 8, 12, 18 and 24 hours respectively, recording mass spectrograms, and the solution stability test results are shown in table 1.
Table 1: methodology solution stability examination results
As can be seen from Table 1:
for genotoxic impurity A, the limit of quantitation was 1.56ng/mL, corresponding to 0.78 ppm; the system applicability RSD is 1.9 percent (less than or equal to 15.0 percent), and the system applicability is good; the linear correlation coefficient is 1.000 (more than or equal to 0.990), the intercept deviation is 2 percent (less than or equal to 25 percent), and the linear relation is good in the range of 2 ng/mL-16 ng/mL; the recovery rates of 50%, 100% and 150% of standard addition are all within 15.0%, and the accuracy is good; the solution was stable within 15 hours.
The limit of quantitation for genotoxic impurity B was 1.56ng/mL, corresponding to 0.78 ppm; the system applicability RSD is 2.3 percent (less than or equal to 15.0 percent), and the system applicability is good; the linear correlation coefficient is 0.997 (more than or equal to 0.990), the intercept deviation is 3 percent (less than or equal to 25 percent), and the linear relation is good in the range of 2 ng/mL-16 ng/mL; the recovery rates of 50%, 100% and 150% of standard addition are all within 15.0%, and the accuracy is good; the solution was stable within 15 hours.
The limit of quantitation for genotoxic impurity C is 1.56ng/mL, corresponding to 0.78 ppm; the system applicability RSD is 0.9 percent (less than or equal to 15.0 percent), and the system applicability is good; the linear correlation coefficient is 0.995 (more than or equal to 0.990), the intercept deviation is 18 percent (less than or equal to 25 percent), and the linear relation is good in the range of 2 ng/mL-16 ng/mL; the recovery rates of 50%, 100% and 150% of standard addition are all within 15.0%, and the accuracy is good; the solution was stable within 15 hours.
The method for detecting the genotoxic impurities in the varenicline intermediate can effectively separate the main component peak and the genotoxic impurity peak of varenicline, greatly improves the detection sensitivity of genotoxic impurities, and can simply, quickly and stably detect the genotoxic impurities in the varenicline intermediate, so that the quality of the varenicline intermediate is effectively controlled, and the risk possibly brought by undetected harmful impurities is avoided.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains to private information, several simple deductions or substitutions may be made without departing from the spirit of the invention, and all shall be considered as belonging to the scope of the invention.
Claims (10)
1. A method for detecting nitrosamine genotoxic impurities in varenicline intermediate comprises the step of measuring the content of nitrosamine genotoxic impurities by adopting a high-resolution liquid chromatography and mass spectrometry combined technology.
2. The detection method according to claim 1, characterized in that: the genotoxic impurity is compound A, B, C, and the chemical structural formulas are respectively as follows:
the varenicline intermediate has the following structure:
or preferably the use of the detection method as claimed in claim 1 for the detection of genotoxic impurities A or B or C in varenicline intermediates.
3. The detection method according to claim 1 or 2, characterized in that: the detection method adopts high resolution liquid chromatography and mass spectrometry combined technology for determination, and comprises the following chromatographic and mass spectrometry conditions:
(1) chromatographic conditions
The filler is an octadecyl bonded silica gel chromatographic column,
the mobile phase is 0.1 percent formic acid water solution and 0.1 percent formic acid acetonitrile solution with a certain volume ratio, gradient elution is carried out,
the column temperature is 40-50 ℃,
the flow rate of the mobile phase is 0.4-0.6 mL/min,
the detection wavelength is 210-230 nm;
(2) conditions of Mass Spectrometry
The ion source is a heatable electrospray ionization source, the temperature of the ion source is 400-500 ℃,
the sheath gas flow rate is 50 to 60arbitrary units, the auxiliary gas flow rate is 10 to 20arbitrary units,
the collision energy is 50 to 70NCE,
the temperature of the ion transmission tube is 300-400 ℃,
the scan mode is a positive ion mode,
the collection mode is a parallel reaction monitoring mode or a selective ion monitoring mass spectrometry mode.
4. The detection method according to claim 3, characterized in that: the detection method adopts high resolution liquid chromatography and mass spectrometry combined technology for determination, and comprises the following sample preparation method and detection method:
(3) sample preparation method
Preparing a reference substance solution: respectively precisely weighing an appropriate amount of A, B, C genotoxic impurity reference substance, dissolving with acetonitrile water solution at a volume ratio of 50:50, and respectively preparing reference substance solution containing 7.8ng of genotoxic impurity A, B, C per 1 mL;
preparing a test solution: accurately weighing varenicline intermediate, and preparing a solution containing varenicline with the concentration of 2mg/mL by using an acetonitrile water solution with the volume ratio of 50: 50;
(4) detection method
Injecting the test solution and each reference solution into a high-resolution liquid chromatograph-mass spectrometer for detection.
5. The detection method according to claim 3, characterized in that: in the chromatographic conditions in the step (1), the octadecyl bonded silica gel chromatographic column is an Agilent _ ZORBAX Eclipse Plus C18_150x4.6mm _3.5 μm type column.
6. The detection method according to claim 3, characterized in that: in the chromatographic condition of the step (1), the mobile phases of gradient elution are 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution with different volume ratios, and the following elution gradient is adopted:
The above percentages are by volume.
7. The detection method according to claim 3, characterized in that: in the chromatographic conditions of the step (1), the column temperature of a chromatographic column is set to be 45 ℃;
or preferably, the flow rate of the mobile phase in the chromatographic conditions of the step (1) is 0.5 mL/min;
or preferably, in the chromatographic condition of the step (1), the detection wavelength is 214 nm.
8. The detection method according to claim 3, characterized in that: in the mass spectrum condition of the step (2), the temperature of an electrospray ionization source ion source is 420-480 ℃;
or preferably, in the mass spectrum condition of the step (2), the flow rate of the sheath gas of the high-resolution mass spectrum is 55arbitrary units, and the auxiliary gas flow rate is 15arbitrary units;
or preferably, in the mass spectrum condition of the step (2), the temperature of the ion transmission tube is 360-390 ℃.
9. The detection method according to claim 4, characterized in that: in the sample preparation method in the step (3), the sample solution is prepared by taking varenicline intermediate, and mixing the varenicline intermediate with water and acetonitrile in a volume ratio of 50: preparing a solution containing 2mg/mL varenicline from the mixed solution of 50;
or preferably, in the detection method in the step (4), the sample solution and each reference solution are injected into a high-resolution liquid chromatograph-mass spectrometer, about 1mL of the sample solution is filled in a 1.5mL sample injection vial, and a sample is collected and a mass spectrogram is recorded through an automatic sample injection device of a high performance liquid chromatograph.
10. The detection method according to one of claims 2 to 9, characterized in that: the content of A in the nitrosamine genotoxic impurity in the varenicline intermediate formula I is less than or equal to 3.9ppm, the content of B is less than or equal to 3.9ppm and the content of C is less than or equal to 3.9 ppm; more preferably, the A content is less than or equal to 0.78ppm, the B content is less than or equal to 0.78ppm and the C content is less than or equal to 0.78 ppm;
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