CN114088843B - Detection method for nitrosamine genotoxic impurities in valance intermediate - Google Patents
Detection method for nitrosamine genotoxic impurities in valance intermediate Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 79
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 69
- 238000001514 detection method Methods 0.000 title claims abstract description 68
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 67
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 239000000543 intermediate Substances 0.000 claims abstract description 47
- 229960004751 varenicline Drugs 0.000 claims abstract description 31
- JQSHBVHOMNKWFT-DTORHVGOSA-N varenicline Chemical compound C12=CC3=NC=CN=C3C=C2[C@H]2C[C@@H]1CNC2 JQSHBVHOMNKWFT-DTORHVGOSA-N 0.000 claims abstract description 30
- 238000011141 high resolution liquid chromatography Methods 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 5
- 238000005516 engineering process Methods 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims abstract description 4
- 231100000331 toxic Toxicity 0.000 claims abstract description 3
- 230000002588 toxic effect Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 39
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- 238000001819 mass spectrum Methods 0.000 claims description 16
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 14
- 239000013558 reference substance Substances 0.000 claims description 13
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 239000000523 sample Substances 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
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- 238000004896 high resolution mass spectrometry Methods 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 8
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- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 4
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- ZXVONLUNISGICL-UHFFFAOYSA-N 4,6-dinitro-o-cresol Chemical group CC1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O ZXVONLUNISGICL-UHFFFAOYSA-N 0.000 description 1
- 206010017472 Fumbling Diseases 0.000 description 1
- 206010057852 Nicotine dependence Diseases 0.000 description 1
- 208000025569 Tobacco Use disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 229940059344 chantix Drugs 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
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- 239000003480 eluent Substances 0.000 description 1
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- 125000000018 nitroso group Chemical group N(=O)* 0.000 description 1
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- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 1
- 229910052611 pyroxene Inorganic materials 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- TWYFGYXQSYOKLK-CYUSMAIQSA-N varenicline tartrate Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O.C12=CC3=NC=CN=C3C=C2[C@H]2C[C@@H]1CNC2 TWYFGYXQSYOKLK-CYUSMAIQSA-N 0.000 description 1
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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
- G01N30/02—Column chromatography
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- 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
- G01N30/02—Column chromatography
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- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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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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Abstract
The invention provides a detection method of nitrosamine genotoxic impurities in varenicline intermediates, which comprises the steps of adopting high-resolution liquid chromatography and mass spectrometryThe content of nitrosamine gene impurities is determined by spectrum combination technology, the toxic impurities of the gene are compounds A, B, C, and the chemical structural formulas are as follows:the valanserine intermediate structure is as follows:
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a detection method of nitrosamine genotoxic impurities in varenicline intermediates.
Background
Varenicline is a drug developed by the american-type sciences company for the treatment of nicotine addiction. There are numerous reports on the preparation and synthesis of valanserine intermediates at present, but there is less concern about genotoxic impurities.
In original patent CN101410110a, valanserine may be present in one or more of several mononitro, monoamino, mixed amino nitro, diamino or dinitro intermediate impurities, but no study was made of nitroso impurities.
For 6 months 2021, the production of the drug for stopping smoking, chantix (varenicline), N-nitroso-varenicline, was stopped by the pyroxene due to unacceptable nitrosamine content. Currently, genotoxicity detection in varenicline is of increasing interest. Therefore, a detection method for genotoxic impurities in the drug synthesis process, which has high sensitivity and good specificity, is developed, particularly, the intermediate nitrosamine genotoxic impurities are strictly controlled, and the accurate and effective control of the quality of varenicline drugs can be realized, so that the safety of the varenicline patients in drug administration is ensured, and the detection method has great research significance.
The invention comprises the following steps:
in order to solve the problems of scarcity of a detection method of genotoxic impurities of varenicline intermediates and safety of valenicline medication. The invention provides a detection method of nitrosamine genotoxic impurities in varenicline intermediate, which is shown in the formula I, and has the chemical name of 2,3,4, 5-tetrahydro-7-nitro-3- (trifluoroacetyl) -1, 5-methyl bridge-1-hydrogen-3-benzonitrogenThe structure is as follows:
the invention provides a detection method of nitrosamine gene toxic impurities in varenicline intermediates, which adopts high-resolution liquid chromatography and mass spectrometry technology to detect nitrosamine gene impurities.
Further, according to the detection method of nitrosamine genotoxic impurities in varenicline intermediates, the genotoxic impurities are compounds A, B, C, and the chemical structural formulas of the genotoxic impurities are as follows:
the invention provides a detection method for nitrosamine genotoxic impurities in varenicline intermediates, which adopts a high-resolution liquid chromatography and mass spectrometry combined technology for determination and comprises the following detection methods:
(1) Chromatographic conditions
The filler is octadecyl bonded silica gel chromatographic column,
the mobile phase is 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution with a certain volume ratio, the 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) Mass spectrometry conditions
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 50-60 arbitrary units, the auxiliary gas flow rate is 10-20 arbitrary units,
collision energy of 50 to 70 (NCE)
The temperature of the ion transmission tube is 300-400 ℃,
the scan mode is a positive ion mode and,
the acquisition mode is a parallel reaction monitoring mode or a selective ion monitoring mass spectrometry mode.
Further, the detection method of nitrosamine genotoxic impurities in varenicline intermediates provided by the invention comprises the following sample preparation method:
(3) Sample preparation method
Preparing a reference substance solution: preparing a control solution with 7.8ng of genotoxic impurities A, B, C per 1 mL;
preparing a test solution: the varenicline intermediate is precisely weighed and prepared into a solution with the concentration of 2mg/mL of varenicline by using an aqueous solution of acetonitrile (50:50, v/v).
Further, the detection method of nitrosamine genotoxic impurities in varenicline intermediates provided by the invention comprises the following detection methods:
(4) Detection method
And (3) injecting the sample solution and each reference substance solution into a high-resolution liquid chromatography-mass spectrometer for detection.
Furthermore, in the detection method of nitrosamine genotoxic impurities in varenicline intermediates, in the chromatographic condition of the step (1), the model of an octadecyl bonded silica gel chromatographic column is Agilent_ ZORBAX Eclipse Plus C18_150x4.6mm_3.5 μm.
In the detection method of nitrosamine genotoxic impurities in varenicline intermediates, in the chromatographic condition in 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:
| time (min) | 0.1% formic acid aqueous 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 percentages are by volume.
Further, according to the detection method of nitrosamine genotoxic impurities in valance intermediate, in the chromatographic condition of step (1), the column temperature of the chromatographic column is set to be 45 ℃.
Furthermore, according to the detection method of nitrosamine genotoxic impurities in the valanserine intermediate, in the chromatographic condition of the step (1), the flow rate of a mobile phase is 0.5mL/min.
Furthermore, in the detection method of nitrosamine genotoxic impurities in varenicline intermediates, in the chromatographic condition in the step (1), the detection wavelength is 214nm.
Further, according to the detection method of nitrosamine genotoxic impurities in varenicline intermediates, in the mass spectrum condition of the step (2), the temperature of an ion source of an electrospray ionization source is 420-480 ℃.
In the detection method of nitrosamine genotoxic impurities in valance intermediate, in the mass spectrum condition of step (2), the high-resolution mass spectrum sheath air flow rate is 55 arbitrary units, and the auxiliary air flow rate is 15 arbitrary units.
Further, according to the detection method of nitrosamine genotoxic impurities in valance intermediate, in the mass spectrum condition of the step (2), the temperature of the ion transmission tube is 360-390 ℃.
In the method for detecting nitrosamine genotoxic impurities in varenicline intermediates, in the step (3), sample solution is prepared by taking varenicline intermediates, and the volume ratio of water to acetonitrile is 50:50 was formulated as a solution containing 2mg/mL varenicline.
In the detection method of step (4), the sample solution and each reference solution are injected into a high-resolution liquid chromatography-mass spectrometer, about 1mL of the sample solution is taken from a 1.5mL sample injection vial, and the sample is collected and a mass spectrogram is recorded by an automatic sample injection device of a high-performance liquid chromatograph. The detection method of the nitrosamine genotoxic impurities in the valaciclolin intermediate can well control the nitrosamine genotoxic impurities in the valaciclolin intermediate formula I, wherein the content (or limit) of A 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 3.9ppm; 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 0.78ppm.
Compared with the prior art, the invention has better beneficial effects:
(1) The detection method of nitrosamine genotoxic impurities in the valance intermediate provided by the invention can effectively separate the main component peak and the genotoxic impurity peak of the valance intermediate, and can provide higher specificity at the same time, avoid detection of false positive impurities.
(2) The invention detects genotoxic impurities in varenicline intermediate by high resolution liquid chromatography-mass spectrometry, and adopts octadecylsilane chemically bonded silica gel as filler.
(3) According to the detection method, the ratio of the mobile phase has great influence on the separation degree of the genotoxic impurities and the valance in the experimental result, further research shows that the accurate separation of the genotoxic impurities and the valance intermediate can be achieved by setting mobile phases with different ratios for gradient elution, and finally, the condition that the ratio of the mobile phase to 0.1% formic acid aqueous solution to 0.1% formic acid acetonitrile solution is 50 is confirmed within 0 to 8 minutes: 50-35: 65, in 8 to 10 minutes, in a ratio of 35:65, hold for 2 minutes, 0.1% acetonitrile formate solution from 65% to 100% over 10 to 15 minutes, then adjust the eluent ratio back to 50:50.
(4) According to the detection method, the collision energy of target ions has a great influence on the sensitivity of genotoxic impurities in detection results, through research, through a high-resolution mass spectrum collision chamber, it is found that the compound A has the maximum ion response for quantifying the mass-to-charge ratio (189.0788) when the collision energy is 60 (NCE), the compound B has the maximum ion response for quantifying the mass-to-charge ratio (156.0808) when the collision energy is 50 (NCE), the compound C has the maximum ion response for quantifying the mass-to-charge ratio (128.0628) when the collision energy is 70 (NCE), and finally, the sample injection amount is 15 mu L, the compound A has the collision energy of 60 (NCE), the quantified mass-to-charge ratio is 189.0788, the compound B has the collision energy of 50 (NCE), the quantified mass-to-charge ratio is 156.0808, the C has the collision energy of 70 (NCE), and the quantified 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 valance intermediate, reduce the occurrence of side effects of patient medication, and ensure the medication safety of the patient to a certain extent.
Drawings
Fig. 1, A, B, C, are liquid phase-high resolution mass spectrometry overlays of three genotoxicities.
FIG. 2 blank for liquid-high resolution mass spectrum of genotoxic impurity A
FIG. 3 liquid-high resolution mass spectrum of genotoxic impurity A
FIG. 4 liquid phase-high resolution mass spectrum of genotoxic impurity B blank spectra
FIG. 5 liquid-high resolution mass spectrum of genotoxic impurity B
FIG. 6 blank for liquid-high resolution mass spectrometry of genotoxic impurity C
FIG. 7 liquid-high resolution mass spectrum of genotoxic impurity C
Detailed Description
The acetonitrile in the mobile phase of the high performance liquid phase in the examples below is chromatographically pure acetonitrile. The temperature in the embodiment is set temperature, the allowable temperature error of the high-performance liquid module is +/-2 ℃, and the allowable temperature error of the high-resolution mass spectrum module is +/-5 ℃.
The detection method is an external standard method in the field of drug analysis, and the following experimental processes and operation modes in the experimental methods 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 fumbling
(1) Chromatographic conditions
The filler is octadecyl bonded silica gel chromatographic column,
the mobile phases are 0.1% formic acid aqueous solution and 0.1% 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 214nm.
(2) Mass spectrometry conditions
The ion source is a heatable electrospray ionization source, and the temperature of the ion source is 450 DEG C
The sheath gas flow rate is 55 arbitrary units, the auxiliary gas flow rate is 15 arbitrary units,
collision energy of 50 to 70 (NCE)
The ion transport tube temperature was 380 c,
the scan mode is a positive ion mode and,
the acquisition mode is a parallel reaction monitoring mode.
(3) Sample preparation
Preparing a reference substance solution: the A, B, C genotoxic impurity control is precisely weighed and dissolved by acetonitrile water solution (50:50, v/v) to prepare a control solution with 7.8ng of genotoxic impurity A, B, C in each 1 mL.
Preparing a test solution: the varenicline intermediate is precisely weighed and prepared into a solution with the concentration of 2mg/mL of the varenicline intermediate by using an aqueous solution of acetonitrile (50:50, v/v).
(4) Detection method
And (3) injecting the sample solution and each reference substance solution into a high-resolution liquid chromatography-mass spectrometer.
Experimental results:
chromatogram results: the chromatographic comparison chart of A, B, C genotoxic impurities, the chromatogram of blank solution and the chromatograms of the solutions of the various controls are shown in figures 1-7. Three genotoxic impurities obtained according to the above chromatographic conditions can be effectively separated.
According to analysis of the impurity reference substance in the valance intermediate, the impurity peak in the valance intermediate can be determined to be A, B, C genotoxic impurity.
Example 2 methodology investigation-System applicability
According to the expert model and the reasoning of software, the genotoxic impurities A, B, C are all 3 types of impurities in ICH, and according to the guiding principle of ICH M7, the impurities are required to be researched and controlled. Comprehensive Toxicology Threshold (TTC) 1.5 mug/day data and actual process conditions, and finally confirm that the limits of genotoxic impurities A, B, C are 3.9ppm. According to the limit and the solubility of the valance intermediate, 1mL of control solution containing 7.8ng of genotoxic impurities A, B, C is prepared respectively, 6 needles are repeatedly injected for balancing the system, a mass spectrum is recorded, and the system applicability test results are shown in Table 1.
EXAMPLE 3 methodology investigation-quantitative limitation
According to the control solution prepared in example 1, diluted 5 times, a quantitative limit solution containing 7.8ng of genotoxic impurity A, 7.8ng of genotoxic impurity B and 7.8ng of genotoxic impurity C was prepared, the signal to noise ratio of each impurity was more than 10, and the quantitative limit test results are shown in Table 1.
EXAMPLE 4 methodology investigation-linearity and Range
The three genotoxic impurities are weighed respectively, the reference substances are right amount, dissolved by a mixed solvent of water and acetonitrile and diluted to proper concentration, the solution is taken for detection, and the linear and range test results are shown in table 1.
Example 5 methodology investigation-accuracy
Weighing a proper amount of varenicline intermediate, adding a proper amount of reference stock solution to obtain standard solutions with the addition of 50%, 100% and 150% of each impurity, feeding the solutions into 3 needles in parallel at each concentration, recording mass spectrograms, and testing the accuracy results shown in Table 1.
EXAMPLE 6 methodology investigation of solution stability
Taking a control solution under the system applicability item, standing at room temperature, respectively carrying out sample injection test at 0, 2, 4, 8, 12, 18 and 24 hours, recording a mass spectrum, and carrying out solution stability test results shown in table 1.
Table 1: methodology solution stability investigation results
As can be seen from table 1:
for genotoxic impurity A, the limit of quantification is 1.56ng/mL, corresponding to 0.78ppm; 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 standard adding recovery rate of 50%, 100% and 150% is within 15.0%, and the accuracy is good; the solution was stable over 15 hours.
The quantitative limit for the genotoxic impurity B is 1.56ng/mL, which is equivalent to 0.78ppm; 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 standard adding recovery rate of 50%, 100% and 150% is within 15.0%, and the accuracy is good; the solution was stable over 15 hours.
The quantitative limit of the genotoxic impurity C is 1.56ng/mL, which is equivalent to 0.78ppm; 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 standard adding recovery rate of 50%, 100% and 150% is within 15.0%, and the accuracy is good; the solution was stable over 15 hours.
The detection method of the genotoxic impurities in the valance intermediate can effectively separate the valance Lin Zhu component peak from the genotoxic impurity peak, and greatly improves the detection sensitivity of the genotoxic impurities, so that the genotoxic impurities in the valance intermediate can be simply, quickly and stably detected, the quality of the valance intermediate can be effectively controlled, and the risk possibly caused by undetected harmful impurities is avoided.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. And a plurality of simple deductions or substitutions can be made by those skilled in the art without departing from the concept of the invention, and the invention is considered to be within the protection scope of the invention.
Claims (12)
1. A detection method of nitrosamine gene toxic impurities in varenicline intermediates comprises the steps of measuring the content of nitrosamine gene impurities by adopting high-resolution liquid chromatography and mass spectrometry, wherein the genotoxic impurities are compounds A, B, C, and the chemical structural formulas of the genotoxic impurities are as follows:
in vareniclineThe structure of the intermediate is as follows:
i is a kind of
The detection method adopts a high-resolution liquid chromatography and mass spectrometry combined technology for detection, and comprises the following chromatographic and mass spectrometry conditions:
(1) Chromatographic conditions
The chromatographic conditions used were as follows:
the filler is octadecyl bonded silica gel chromatographic column,
the mobile phase is 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution with a certain volume ratio, the 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) Mass spectrometry conditions
The ion source is a heatable electrospray ionization source, the temperature of the ion source is 400-500 ℃,
the flow rate of sheath gas is 50-60 arbitrary units, the auxiliary air flow rate is 10-20 arbitrary units,
the collision energy NCE is 50-70,
the temperature of the ion transmission tube is 300-400 ℃,
the scan mode is a positive ion mode and,
the acquisition mode is a parallel reaction monitoring mode or a selective ion monitoring mass spectrometry mode;
the following elution gradient was used:
The percentages are by volume.
2. Use of the assay as claimed in claim 1 for the detection of genotoxic impurity a or B or C in valance intermediates.
3. The detection method according to claim 2, wherein: the detection method adopts a high-resolution liquid chromatography and mass spectrometry combined technology for detection, and comprises the following sample preparation method and detection method:
(3) Sample preparation method
Preparing a reference substance solution: accurately weighing a proper amount of A, B, C genotoxic impurity reference substances respectively, and dissolving the reference substances by using an acetonitrile water solution in a volume ratio of 50:50 to prepare reference substance solutions with 7.8ng of genotoxic impurities A, B, C in each 1mL of reference substance solutions respectively;
preparing a test solution: precisely weighing varenicline intermediate, and preparing a solution containing varenicline with the concentration of 2mg/mL by using an aqueous solution of acetonitrile in a volume ratio of 50:50;
(4) Detection method
And (3) injecting the sample solution and each reference substance solution into a high-resolution liquid chromatography-mass spectrometer for detection.
4. A detection method according to claim 3, wherein: in the chromatographic condition of the step (1), the octadecyl bonded silica gel chromatographic column is an Agilent_ ZORBAX Eclipse Plus C18_150x4.6mm_3.5 μm model column.
5. The detection method according to claim 2, wherein: in the chromatographic condition of the step (1), the column temperature of the chromatographic column is set to be 45 ℃.
6. The detection method according to claim 2, wherein: in the chromatographic conditions of step (1), the flow rate of the mobile phase was 0.5. 0.5mL/min.
7. The detection method according to claim 2, wherein: in the chromatographic condition of the step (1), the detection wavelength is 214nm.
8. The detection method according to claim 2, wherein: in the mass spectrum condition of the step (2), the temperature of an ion source of the electrospray ionization source is 420-480 ℃.
9. The detection method according to claim 2, wherein: in the mass spectrometry condition of the step (2), the high-resolution mass spectrometry sheath gas flow rate is 55 arbitrary units, and the auxiliary gas flow rate is 15 arbitrary units.
10. The detection method according to claim 2, wherein: in the mass spectrum condition of the step (2), the temperature of the ion transmission tube is 360-390 ℃.
11. A detection method according to claim 3, wherein: in the detection method of the step (4), the sample solution to be detected and each reference substance solution are injected into a high-resolution liquid chromatography-mass spectrometer, about 1mL of the sample solution to be detected is taken by a 1.5mL sample injection small bottle, and the sample is collected and a mass spectrum is recorded by an automatic sample injection device of the high-performance liquid chromatograph.
12. The detection method according to one of claims 3 to 11, characterized in that: nitrosamine genotoxic impurities in the valance intermediate formula I are less than or equal to 3.9ppm, less than or equal to 3.9ppm and less than or equal to 3.9ppm;
formula I.
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