CN111060616A - Detection method for nitrosamine impurities in valsartan preparation - Google Patents
Detection method for nitrosamine impurities in valsartan preparation Download PDFInfo
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- CN111060616A CN111060616A CN201911299172.2A CN201911299172A CN111060616A CN 111060616 A CN111060616 A CN 111060616A CN 201911299172 A CN201911299172 A CN 201911299172A CN 111060616 A CN111060616 A CN 111060616A
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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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- 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
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- 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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- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention relates to the technical field of chemical detection and analysis, and particularly relates to a detection method for nitrosamine impurities in a valsartan preparation. A method for detecting nitrosamine impurities in a valsartan preparation is characterized by comprising the following steps: (1) sample treatment: adding a sample into a solvent, performing ultrasonic treatment and centrifugation, and taking supernatant to obtain a solution to be detected; (2) solution preparation: preparing a standard solution; (3) and (3) detection: and (3) detecting and analyzing the solutions obtained in the steps (1) and (2) by adopting liquid chromatography and mass spectrometry. The detection method for nitrosamine impurities in the valsartan preparation provided by the invention has the advantages of high accuracy, good reproducibility and high sensitivity.
Description
Technical Field
The invention relates to the technical field of chemical detection and analysis, and particularly relates to a detection method for nitrosamine impurities in a valsartan preparation.
Background
Nitrosamines are strong carcinogens, one of the most important chemical carcinogens, and one of four major food contaminants. Nitrosamines are contained in foods, cosmetics, beer and cigarettes.
At present, the detection method of nitrosamine impurities mainly comprises a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method and a gas chromatography-thermal energy analyzer method, is mainly applied to the industries of food, drinking water and cosmetics, and has few research reports on the detection method of nitrosamine impurities in medicines at home and abroad. In addition, in the existing detection method, it is often difficult to simultaneously meet the requirements in the aspects of repeatability, accuracy, linearity, sensitivity and the like.
Therefore, the invention aims to provide the method for detecting the nitrosamine impurities in the valsartan preparation, which has the advantages of high accuracy, good repeatability, high sensitivity and the like.
Disclosure of Invention
In order to solve the problems, the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which comprises the following steps:
(1) sample treatment: adding a sample into a solvent, performing ultrasonic treatment and centrifugation, and taking supernatant to obtain a solution to be detected;
(2) solution preparation: preparing a standard solution;
(3) and (3) detection: and (3) detecting and analyzing the solutions obtained in the steps (1) and (2) by adopting liquid chromatography and mass spectrometry.
As a preferable technical scheme, in the step (1), the solvent is selected from one or more of dichloromethane, methanol, acetonitrile, ethyl acetate, ethanol and n-hexane.
As a preferable technical scheme, in the solvent, the volume ratio of dichloromethane to methanol is (1-3): 1.
As a preferable technical scheme, in the step (1), the average particle size of the sample is 200-230 meshes.
In a preferable embodiment, in step (3), the mobile phase a of the liquid chromatography is an aqueous acetic acid solution.
As a preferable technical scheme, in the step (3), the mobile phase B phase of the liquid chromatogram is a mixture of formic acid and methanol.
As a preferable technical solution, in the step (3), the gradient elution conditions of the liquid chromatography are as follows:
0-3 min: mobile phase a 98% → 30%, mobile phase B2% → 70%;
3-3.2 min: mobile phase a 30% → 28%, mobile phase B70% → 72%;
3.2-4 min: mobile phase a is 28% → 28%, mobile phase B is 72% → 72%;
4-4.2 min: mobile phase a is 28% → 0%, mobile phase B is 72% → 100%;
4.2-5.5 min: mobile phase A is 0% → 0%, mobile phase B is 100% → 100%;
5.5-5.6 min: mobile phase a is 0% → 98%, mobile phase B is 100% → 2%;
5.6-8 min: mobile phase a is 98% → 98%, and mobile phase B is 2% → 2%.
As a preferable technical scheme, in the step (3), the ion source type of the mass spectrum is an electric bombardment ion source.
As a preferred technical scheme, the parameters of the electric bombardment ion source of the mass spectrum are as follows: electron energy: 50eV, filament emission current: 50-80 muA.
As a preferred technical scheme, the mass spectrum analysis adopts an MRM mode.
Has the advantages that: according to the detection method for nitrosamine impurities in the valsartan preparation, the valsartan preparation is treated by adopting a proper solvent, so that the accuracy of the detection method is improved; the accuracy and the sensitivity of detection are improved by elaborately setting liquid chromatography conditions, mass spectrum parameters and the like; meanwhile, due to the mutual cooperation among the special treatment of the sample, the setting of the liquid chromatography condition and the setting of the mass spectrum parameters, the detection method for the nitrosamine impurities in the valsartan preparation, provided by the invention, has the advantages of high accuracy, good repeatability and high sensitivity.
Detailed Description
The technical features of the technical solutions provided by the present invention are further clearly and completely described below with reference to the specific embodiments, and the scope of protection is not limited thereto. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The words "preferred", "more preferred", and the like, in the present invention refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
The invention provides a detection method for nitrosamine impurities in a valsartan preparation, which comprises the following steps:
(1) sample treatment: adding a sample into a solvent, performing ultrasonic treatment and centrifugation, and taking supernatant to obtain a solution to be detected;
(2) solution preparation: preparing a standard solution;
(3) and (3) detection: and (3) detecting and analyzing the solutions obtained in the steps (1) and (2) by adopting liquid chromatography and mass spectrometry.
Nitrosamine impurities in the valsartan preparation are N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA).
The CAS number of N-nitrosodimethylamine is 62-75-9.
The CAS number of the N-nitrosodiethylamine is 55-18-5.
The valsartan preparation contains not only valsartan which is a main active ingredient, but also a plurality of auxiliary ingredients such as microcrystalline cellulose, crospovidone, povidone, sodium dodecyl sulfate, magnesium stearate, gelatin, sodium dodecyl sulfate, iron oxide, titanium dioxide and the like, so that dissolution and extraction of a target ingredient are interfered through the combined action of a plurality of mechanisms such as physics, chemistry and the like in the extraction process, and the accuracy of the detection method is influenced. Effective pre-treatment of valsartan formulations is required for more accurate identification and analysis of NDMA and NDEA in valsartan formulations.
Sample processing
The sample treatment of the invention comprises the following steps: and adding a solvent into the sample, performing ultrasonic treatment and centrifugation, and taking supernatant to obtain the solution to be detected.
The sample is a valsartan formulation, and the present invention does not specifically limit the sample.
In one embodiment, the solvent is selected from one or more of dichloromethane, methanol, acetonitrile, ethyl acetate, ethanol, n-hexane.
In a preferred embodiment, the solvent is dichloromethane, methanol.
In one embodiment, the volume ratio of dichloromethane to methanol is (1-3): 1.
in a preferred embodiment, the volume ratio of dichloromethane to methanol is 2: 1.
in one embodiment, the average particle size of the sample is 200-230 mesh.
In a preferred embodiment, the sample has an average particle size of 220 mesh.
In one embodiment, the sonication time is 10-20 min.
In a preferred embodiment, the sonication time is 15 min.
In one embodiment, the sonication temperature is 37-40 ℃.
In a preferred embodiment, the sonication temperature is 38 ℃.
In one embodiment, the mass to volume ratio of sample to solvent is (0.8-1.2): 1 mg/mL.
In a preferred embodiment, the mass to volume ratio of sample to solvent is 1: 1 mg/mL.
The inventor finds that the ratio of the dichloromethane to the methanol is (1-3): 1, the accuracy of the detection method can be improved when the ultrasonic temperature is 37-40 ℃. Probably due to the system formed by dichloromethane and methanol, at 37-40 ℃, a plurality of cavities are formed, and the cavities are rapidly expanded and closed, and instantaneous pressure of thousands of atmospheres is generated during closing, so that violent impact action occurs among liquid particles. Solvent molecules overcome the attraction among surrounding molecules and enter the medicine, and simultaneously weaken the interaction force of other auxiliary components (such as gelatin) with NDMA and NDEA, so that the extraction efficiency of NDMA and NDEA is improved, and the accuracy of the detection method is improved.
Further, the applicant found that when the powder particle size or the ultrasonic time is not properly controlled, the accuracy of the detection method is rather lowered because, on the one hand, when the powder particle size is too large, the dissolution and extraction of the target component are reduced due to the degree of destruction or poor dispersing ability; on the other hand, when the particle diameter of the powdery material is too small, the extraction rate of the objective component is lowered due to the increased interaction resulting from the re-entanglement of the macromolecular chains in the system.
Solution preparation
External standard working fluid
In a preferred embodiment, the preparation of the external standard working fluid comprises the following steps: N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) were prepared into external standard working solutions with methanol, respectively, at concentrations of 200. mu.g/mL.
Standard solution
In a preferred embodiment, the preparation of the standard solution comprises the following steps: the external standard working solution obtained above is mixed in equal volume, and diluted twice by methanol in turn to prepare series of standard solutions with concentration gradient, which are respectively marked as C1-C10, and are shown in Table 1.
TABLE 1 concentration of each target compound in the standard solution (ng/mL)
The standard solution stock solution refers to a reagent solution with an accurately known concentration, and is prepared for the purpose of facilitating quantitative analysis, and a working curve is drawn or a calculation standard is made using the standard solution so as to perform quantitative analysis on a target compound.
Detection of
Conditions of liquid chromatography
In a preferred embodiment, the liquid chromatography column is of the ACQUITY UPLC T3(1.8 μm. times.2.1 mm. times.50 mm) specification.
In a preferred embodiment, the mobile phase a of the liquid chromatography is an aqueous acetic acid solution.
In a preferred embodiment, the percentage of acetic acid in the aqueous acetic acid solution by volume is 0.05% to 0.2%.
In a more preferred embodiment, the percentage of acetic acid in the aqueous acetic acid solution by volume is 0.1%.
In a preferred embodiment, the mobile phase B of the liquid chromatography is a mixture of formic acid and methanol.
In a preferred embodiment, the formic acid and methanol mixture contains 0.05% to 0.2% formic acid by volume.
In a more preferred embodiment, the formic acid and methanol mixture contains 0.1% formic acid by volume.
In a preferred embodiment, the gradient elution conditions of the liquid chromatography are:
0-3 min: mobile phase a 98% → 30%, mobile phase B2% → 70%;
3-3.2 min: mobile phase a 30% → 28%, mobile phase B70% → 72%;
3.2-4 min: mobile phase a is 28% → 28%, mobile phase B is 72% → 72%;
4-4.2 min: mobile phase a is 28% → 0%, mobile phase B is 72% → 100%;
4.2-5.5 min: mobile phase A is 0% → 0%, mobile phase B is 100% → 100%;
5.5-5.6 min: mobile phase a is 0% → 98%, mobile phase B is 100% → 2%;
5.6-8 min: mobile phase a is 98% → 98%, and mobile phase B is 2% → 2%.
According to the method, an ACQUITY UPLC T3(1.8 mu m multiplied by 2.1mm multiplied by 50mm) chromatographic column is selected, and the filling particles of the chromatographic column are high-strength silica gel particles, so that the chromatographic column can be compatible with 100% aqueous mobile phase, organic molecules with good polar water solubility can be effectively reserved and separated, and the accuracy of the detection method is improved.
However, the inventors have unexpectedly found that mobile phase a is selected to be 0.05% to 0.2% acetic acid/water, mobile phase B is selected to be 0.05% to 0.2% formic acid/methanol; and the volume ratio of the A and the B in different time periods is controlled, and the sensitivity of the detection method can be improved. The suspected reason is that when 0.05% -0.2% acetic acid/water and 0.05% -0.2% formic acid/methanol are selected as mobile phases, on one hand, the selection of a proper mobile phase is beneficial to separating more interference substances to obtain a target compound, so that the recovery rate is improved, and the accuracy of the detection method is further improved; on the other hand, nitrosamines are liable to form [ M + H ] with formic acid, acetic acid, etc. in the mobile phase]+Positive ions, suitable acidity to further promote ionization; secondly, the specific flow matched chromatographic column can greatly improve the separation speed and the separation degree of the target compound and the interfering substance under a certain gradient condition, and particularly can well reduce the influence of NMBA on NDMA, thereby improving the sensitivity of the detection method.
Conditions of Mass Spectrometry
In a preferred embodiment, the ion source type of the mass spectrometer is an electron impact ion source.
In a preferred embodiment, the parameters of the electron impact ion source of the mass spectrum are as follows: electron energy: 50eV, filament emission current: 50-80 muA.
In a more preferred embodiment, the parameters of the electron impact ion source of the mass spectrometer are as follows: electron energy: 50eV, filament emission current: 60 muA.
In a preferred embodiment, the analysis of the mass spectrum uses the MRM mode.
In a preferred embodiment, the parameters of the electric bombardment ion source of the mass spectrum further comprise: ion source temperature: 220 ℃; quadrupole temperature: 150 ℃; transmission line temperature: 230 ℃; and (3) quantifying ions: NDMA: m/z 74, NDEA: 102 m/z; solvent retardation: and 4 min.
The inventor adopts the electron impact ion source (EI) in this application, utilizes the electron of certain energy direct action to the sample molecule, makes its ionization, and efficient, helps the mass spectrometer obtain high sensitivity and high resolution. Through the mutual synergistic effect of the parameters such as the ion source temperature, the quadrupole rod temperature, the transmission line temperature and the like, on one hand, the signal-to-noise ratio of the equipment is improved, the influence of noise is reduced, and the signal has higher stability; on the other hand, the ionization efficiency is improved, and under the condition of proper mass-to-charge ratio, the interference on quantitative ions is minimized, and qualitative and quantitative analysis can be more accurately carried out.
The applicant has found that when the electron energy: 50 eV; filament emission current: at 50-80 muA, the accuracy and repeatability of the detection method can be further improved, presumably because: electrons generated by the filament enter the ionization chamber through the slits on the two sides of the ionization chamber and collide with the substance to be detected in the ionization chamber, and when the energy of the electrons is 50 eV; when the filament emission current is 50-80 muA, the characteristic fragment ion peak with good signal intensity can be obtained, and the matching of the characteristic peak and the target compound is convenient. When ionization energy of 70eV is used, because the ionization potential of the organic compound in the component to be detected is higher than that of the organic compound in the component to be detected, a plurality of molecular ions are further cracked to form generalized fragment ions, and the molecular ions and the fragment ions of various substances have an overlapping phenomenon, so that spectrogram analysis is difficult.
In the testing process, the ion signal is inhibited due to the influence of the complex matrix effect, and a false positive result is often obtained due to the interference of impurities, so that the accuracy of the detection method is reduced. The inventors found that H in chromatographic mobile phase+、-OH-、-COOH-And Cl introduced by solvent-The method has certain influence on ionization of the object to be detected, and for mobile phases of different systems, due to different elution modes and elution conditions, the fragmentation path of the object to be detected and the abundance of each fragment ion have certain difference, so that the detection and analysis of the target compound are influenced to a certain extent. In the application, the inventor designs the substances and the elution conditions of the mobile phase elaborately, and sets the mass spectrum parameters, so that the matrix effect is weakened, the false positive test result can be effectively eliminated, and the accuracy of the detection method is further improved.
Hereinafter, the present invention will be described in more detail by way of examples, but it should be understood that these examples are merely illustrative and not restrictive. In addition, the starting materials used are all commercially available, unless otherwise specified.
Examples
Example 1
Embodiment 1 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, comprising the following steps:
(1) sample treatment: adding a sample into a solvent, performing ultrasonic treatment and centrifugation, and taking supernatant to obtain a solution to be detected;
(2) solution preparation: preparing a standard solution;
(3) and (3) detection: and (3) detecting and analyzing the solutions obtained in the steps (1) and (2) by adopting liquid chromatography and mass spectrometry.
The nitrosamine impurities in the valsartan preparation are N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA).
In the step (1), the solvent is dichloromethane or methanol.
The volume ratio of the dichloromethane to the methanol is 2: 1.
in the step (1), the average particle size of the sample is 220 meshes.
In the step (1), the ultrasonic time is 15 min.
In the step (1), the ultrasonic temperature is 38 ℃.
In the step (1), the mass-to-volume ratio of the sample to the solvent is 1: 1 mg/mL.
In the step (2), the preparation of the external standard working solution comprises the following steps: N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) were prepared into external standard working solutions with methanol, respectively, at concentrations of 200. mu.g/mL.
In the step (2), the preparation of the standard solution comprises the following steps: the external standard working solution obtained above is mixed in equal volume, and diluted twice by methanol in turn to prepare series of standard solutions with concentration gradient, which are respectively marked as C1-C10, and are shown in Table 1.
TABLE 1 concentration of each target compound in the standard solution (ng/mL)
| Target compound | NDMA | NDEA |
| C1 | 200000 | 200000 |
| C2 | 100000 | 100000 |
| C3 | 50000 | 50000 |
| C4 | 25000 | 25000 |
| C5 | 12500 | 12500 |
| C6 | 6250 | 6250 |
| C7 | 3125 | 3125 |
| C8 | 1562.5 | 1562.5 |
| C9 | 781.25 | 781.25 |
| C10 | 390.625 | 390.625 |
In the step (3), the conditions of the liquid chromatography are as follows:
a) the specification of the liquid chromatography column is ACQUITY UPLC T3(1.8 μm. times.2.1 mm. times.50 mm);
b) the mobile phase A phase is acetic acid aqueous solution, and the volume percentage of acetic acid in the acetic acid aqueous solution is 0.1%;
c) the mobile phase B is a mixture of formic acid and methanol, and the volume percentage of formic acid in the mixture of formic acid and methanol is 0.1%;
d) the gradient elution conditions were:
0-3 min: mobile phase a 98% → 30%, mobile phase B2% → 70%;
3-3.2 min: mobile phase a 30% → 28%, mobile phase B70% → 72%;
3.2-4 min: mobile phase a is 28% → 28%, mobile phase B is 72% → 72%;
4-4.2 min: mobile phase a is 28% → 0%, mobile phase B is 72% → 100%;
4.2-5.5 min: mobile phase A is 0% → 0%, mobile phase B is 100% → 100%;
5.5-5.6 min: mobile phase a is 0% → 98%, mobile phase B is 100% → 2%;
5.6-8 min: mobile phase a is 98% → 98%, and mobile phase B is 2% → 2%.
The mass spectrum conditions in the step (3) are as follows: the ion source type is an electric bombardment ion source, and the electron energy is as follows: 50eV, filament emission current: 60 muA, and the analysis of the mass spectrum adopts an MRM mode; ion source temperature: 220 ℃; quadrupole temperature: 150 ℃; transmission line temperature: 230 ℃; and (3) quantifying ions: NDMA: m/z 74, NDEA: 102 m/z; solvent retardation: and 4 min.
Example 2
The embodiment 2 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same way as the embodiment 1, except that in the step (1), the solvent is dichloromethane.
Example 3
The embodiment 3 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same way as the embodiment 1, except that in the step (1), the solvent is methanol.
Example 4
The embodiment 4 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is similar to the embodiment 1, except that in the step (1), the solvent is a mixture of dichloromethane and acetonitrile, and the volume ratio of dichloromethane to acetonitrile is 2: 1.
example 5
The embodiment 5 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is the same as in embodiment 1, except that in the step (1), the solvent is a mixture of methanol and ethyl acetate, and the volume ratio of ethyl acetate to methanol is 2: 1.
example 6
The embodiment 6 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is the same as the embodiment 1 in specific implementation manner, and is characterized in that in the step (1), the solvent is a mixture of acetonitrile and ethyl acetate, and the volume ratio of the acetonitrile to the ethyl acetate is 2: 1.
example 7
The embodiment 7 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same manner as in the embodiment 1, except that in the step (1), the volume ratio of dichloromethane to methanol is 0.5: 1.
example 8
The embodiment 8 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same way as the embodiment 1, and is characterized in that in the step (1), the volume ratio of dichloromethane to methanol is 5: 1.
example 9
The embodiment 9 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same way as the embodiment 1, but the ultrasonic temperature is 30 ℃ in the step (1).
Example 10
The embodiment 10 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same way as the embodiment 1, but the ultrasonic temperature in the step (1) is 45 ℃.
Example 11
Example 11 of the present invention provides a method for detecting nitrosamine impurities in a valsartan formulation, which is performed in the same manner as example 1, except that in step (1), the sample has an average particle size of 150 mesh.
Example 12
Example 12 of the present invention provides a method for detecting nitrosamine impurities in a valsartan formulation, which is performed in the same manner as example 1, except that in step (1), the sample has an average particle size of 300 mesh.
Example 13
The embodiment 13 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is similar to the embodiment 1, except that in the step (3), the mobile phase a is an acetic acid aqueous solution, and the volume percentage of acetic acid in the acetic acid aqueous solution is 0.5%.
Example 14
The embodiment 14 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is similar to the embodiment 1, except that in the step (3), the mobile phase a is an acetic acid aqueous solution, and the volume percentage of acetic acid in the acetic acid aqueous solution is 0.02%.
Example 15
The embodiment 15 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is similar to the embodiment 1, except that in the step (3), the mobile phase B is a mixture of formic acid and methanol, and the volume percentage of formic acid in the mixture of formic acid and methanol is 0.5%.
Example 16
The embodiment 16 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is similar to the embodiment 1, except that in the step (3), the mobile phase B is a mixture of formic acid and methanol, and the volume percentage of formic acid in the mixture of formic acid and methanol is 0.02%.
Example 17
The embodiment 17 of the present invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same manner as in embodiment 1, except that in step (3), the gradient elution conditions of the liquid chromatography are as follows:
0-3.2 min: mobile phase a 98% → 28%, mobile phase B2% → 72%;
3.2-4.2 min: mobile phase a is 28% → 0%, mobile phase B is 72% → 100%;
4.2-5.6 min: mobile phase a is 0% → 98%, mobile phase B is 100% → 2%;
5.6-8 min: mobile phase a is 98% → 98%, and mobile phase B is 2% → 2%.
Example 18
Example 18 of the present invention provides a method for detecting nitrosamine impurities in valsartan formulations, which is implemented in the same manner as example 1, except that in step (3), the ion source type of the mass spectrum is an electron bombardment ion source, and the electron energy is 70 eV.
Example 19
The embodiment 19 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same way as the embodiment 1, and is characterized in that in the step (3), the ion source type of the mass spectrum is an electric bombardment ion source, and the electron energy is 30 eV.
Example 20
The embodiment 20 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is implemented in the same way as the embodiment 1, and is characterized in that in the step (3), the ion source type of the mass spectrum is an electric bombardment ion source, the electron energy is 50eV, and the filament emission current is 40 muA.
Example 21
The embodiment 21 of the invention provides a method for detecting nitrosamine impurities in a valsartan preparation, which is similar to the embodiment 1, and is characterized in that in the step (3), the ion source type of the mass spectrum is an electric bombardment ion source, the electron energy is 50eV, and the filament emission current is 90 muA.
Performance evaluation
1. Quantitative limit detection
Precisely weighing appropriate amounts of NDMA and NDEA, placing in the same measuring flask, dissolving with methanol, diluting, and determining concentration with signal-to-noise ratio of more than 10 times as the limit of quantitation according to the detection methods described in examples 1-17, wherein the average value is obtained by three times of recovery rate determination. The results are shown in Table 2.
TABLE 2 results of limit of quantitation measurements for examples 1-17
2. Accuracy detection
Precisely weighing appropriate amounts of NDMA and NDEA, placing in a same measuring flask, dissolving with methanol, and diluting into solutions with NDMA and NDEA concentrations of 0.1 μ g/ml respectively to obtain reference solution; concentrating 200mL of solution to be detected to 2mL, placing the solution in a 10mL measuring flask, adding 1mL of reference substance solution, dissolving with methanol, diluting to scale, shaking up, detecting and analyzing according to the detection methods described in examples 1-17, calculating recovery rate and RSD, wherein the recovery rate is measured six times and averaged. The results are shown in Table 3.
TABLE 3 results of accuracy tests of examples 1-17
3. Linear detection
According to the method for detecting nitrosamine impurities in the valsartan preparation, which is described in example 1, a standard stock solution is taken for sample injection analysis, and a linear equation and a correlation coefficient are calculated by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate. The results are shown in Table 4.
Table 4 results of linear detection of example 1
| Compound (I) | Linear equation of equations | Correlation coefficient |
| NDMA | y＝6.49*104x+24.68 | R＝0.999 |
| NDEA | y＝2.07*105x+42.23 | R＝0.999 |
The method for detecting nitrosamine impurities in the valsartan preparation, which is described in embodiment 1, can obtain characteristic fragment ion peaks with good signal intensity, and has relatively low noise and relatively accurate baseline; the detection methods for the nitrosamine impurities in the valsartan preparation, which are described in examples 19 and 20, have no obvious mass spectrum peak; the methods for detecting nitrosamine impurities in valsartan formulations described in examples 18 and 21 have relatively high noise and baseline drift.
According to the result, the minimum quantitative concentrations of NDMA and NDEA in the detection method for nitrosamine impurities in the valsartan preparation are both 0.01 mu g/ml, the recovery rate is 99-102%, the standard relative deviation is not more than 4%, and the correlation coefficient is more than 0.99. The method has high accuracy, good reproducibility and high sensitivity.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.
Claims (10)
1. A method for detecting nitrosamine impurities in a valsartan preparation is characterized by comprising the following steps:
(1) sample treatment: adding a sample into a solvent, performing ultrasonic treatment and centrifugation, and taking supernatant to obtain a solution to be detected;
(2) solution preparation: preparing a standard solution;
(3) and (3) detection: and (3) detecting and analyzing the solutions obtained in the steps (1) and (2) by adopting liquid chromatography and mass spectrometry.
2. The method for detecting nitrosamine impurities in a valsartan formulation as claimed in claim 1, wherein in step (1), the solvent is selected from one or more of dichloromethane, methanol, acetonitrile, ethyl acetate, ethanol and n-hexane.
3. The method for detecting nitrosamine impurities in a valsartan formulation as claimed in claim 2, wherein the solvent comprises dichloromethane and methanol at a volume ratio of (1-3): 1.
4. the method for detecting nitrosamine impurities in a valsartan formulation as claimed in claim 1, wherein in step (1), the average particle size of said sample is 200-230 mesh.
5. The method for detecting nitrosamine impurities in a valsartan formulation as claimed in claim 1, wherein in step (3), the mobile phase A of the liquid chromatography is aqueous acetic acid.
6. The method for detecting nitrosamine impurities in a valsartan formulation as claimed in claim 1, wherein in step (3), the mobile phase B of the liquid chromatography is a mixture of formic acid and methanol.
7. The method for detecting nitrosamine impurities in a valsartan formulation as claimed in claim 1, wherein in step (3), the gradient elution conditions of the liquid chromatography are as follows:
0-3 min: mobile phase a 98% → 30%, mobile phase B2% → 70%;
3-3.2 min: mobile phase a 30% → 28%, mobile phase B70% → 72%;
3.2-4 min: mobile phase a is 28% → 28%, mobile phase B is 72% → 72%;
4-4.2 min: mobile phase a is 28% → 0%, mobile phase B is 72% → 100%;
4.2-5.5 min: mobile phase A is 0% → 0%, mobile phase B is 100% → 100%;
5.5-5.6 min: mobile phase a is 0% → 98%, mobile phase B is 100% → 2%;
5.6-8 min: mobile phase a is 98% → 98%, and mobile phase B is 2% → 2%.
8. The method for detecting nitrosamine-type impurities in a valsartan formulation as claimed in claim 1, wherein in step (3), the type of ion source of the mass spectrum is an electron bombardment ion source.
9. The method of claim 8 for detecting nitrosamine-type impurities in a valsartan formulation, wherein the parameters of the source of the electric bombardment ions of the mass spectrum are as follows: electron energy: 50eV, filament emission current: 50-80 muA.
10. The method of claim 1 for the detection of nitrosamine-type impurities in a valsartan formulation, wherein said mass spectrometric analysis employs an MRM mode.
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| CN114397374A (en) * | 2021-12-09 | 2022-04-26 | 石家庄四药有限公司 | Method for detecting N-nitrosodimethylamine and N-nitrosodiethylamine in raw material of entacapone or preparation thereof |
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