CN115598267B - Analysis method of potential genotoxic impurities of glibenclamide Ji Tezhong - Google Patents
Analysis method of potential genotoxic impurities of glibenclamide Ji Tezhong Download PDFInfo
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Abstract
The invention relates to the technical field of medicine analysis, in particular to an electrospray high performance liquid chromatography analysis method of potential genotoxic impurities in glibenches Ji Tezhong, which is used for analysis and detection of N-amino-3-azabicyclo [3, 0] octane as the potential genotoxic impurities in glibenches Ji Tezhong. Firstly, preparing a sample solution by using absolute methanol as a solvent; then, the quaternary ammonium salt strong anion exchange group bonded high-purity silica gel is used as a filler to separate the N-amino-3-azabicyclo [3, 0] octane which is a potential genetic toxic impurity; and finally, adopting an electric fog type detector to analyze and detect, and realizing the control of the potential genetic toxic impurities of the glibenches Ji Tezhong.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to an electrospray high performance liquid chromatography analysis method of potential genotoxic impurities, which is used for analyzing and detecting N-amino-3-azabicyclo [3, 0] octane which is a potential genotoxic impurity of glibenches Ji Tezhong.
Background
Gliclazide (Gliclazide), chemical name 1- (3-azabicyclo [3, 0)]Octyl) -3-p-toluenesulfonyl urea of formula C 12 H 21 N 3 O 3 S, the structural formula is as follows:
gliclazide is a second-generation sulfonylurea oral hypoglycemic agent developed by French Shi Weiya company, has the dual effects of reducing blood sugar and improving blood coagulation, can improve the metabolism of diabetics, can improve or delay the occurrence of diabetic vascular complications, is one of the most commonly used drugs for treating type 2 diabetes at present, and is also a first-line oral hypoglycemic agent in China.
Regarding the synthesis of gliclazide, most of the literature reports that cyclopentane phthalic anhydride is used as a raw material, and is subjected to reflux reaction with hydrazine hydrate in a solvent to obtain N-amino-1, 2-cyclopentane dicarboximide, and then KBH is used for preparing the gliclazide 4 /AlCl 3 Reduction to give N-amino-3-azabicyclo [3, 0]]And finally, condensing octane and hydrochloride thereof with p-toluenesulfonyl urea to obtain gliclazide. The synthetic process route is as follows:
according to the synthetic route, N-amino-3-azabicyclo [3, 0] octane is a key intermediate for synthesizing gliclazide, and amide bonds exist in the molecular structure of gliclazide, so that degradation is easy to occur to generate N-amino-3-azabicyclo [3, 0] octane, and N-amino-3-azabicyclo [3, 0] octane is not only a process impurity of gliclazide but also degradation impurity. In addition, N-amino-3-azabicyclo [3, 0] octane belongs to the class of hydrazines, and is a potentially genotoxic impurity, and whether the content exceeds a limit or not needs to be monitored in gliclazide according to relevant regulations of drug registration rules and ICH M7 guidelines. The control limit of N-amino-3-azabicyclo [3, 0] octane was set to 0.001% based on a maximum daily dose of gliclazide of 120 mg per day, calculated as a limit level of genotoxic impurities of less than 1.5 μg per day.
The early research results show that the N-amino-3-azabicyclo [3, 0] octane does not contain double bonds, conjugate bonds and other ultraviolet absorbing functional groups in the molecular structure, and cannot be quantitatively detected by a liquid chromatography ultraviolet detector; the sensitivity of the universal detector such as the differential refraction detector, the evaporation detector and the like is low, and the detection requirement cannot be met; the mass spectrum detector has higher sensitivity, but the response signal is easily affected by the ionization degree of the compound, has poor stability, is expensive and is not easy to popularize and apply; by adopting a gas chromatographic analysis method, the gliclazide is degraded at high temperature to generate N-amino-3-azabicyclo [3, 0] octane, so that the repeatability and the accuracy of the detection result are poor.
In summary, in order to achieve the structural characteristics and physicochemical properties of gliclazide and N-amino-3-azabicyclo [3, 0] octane and meet the requirement of detection sensitivity, the invention develops an electrospray high performance liquid chromatography analysis method which is used for separating and detecting N-amino-3-azabicyclo [3, 0] octane which is a potential genotoxic impurity of gliclazide Ji Tezhong. At present, no published report exists on a liquid chromatography determination method of genotoxic impurities in gliclazide, so that the method has extremely important significance for quality control of gliclazide bulk drugs and medication safety assurance of preparations thereof.
Disclosure of Invention
Aiming at the problem that high-end detection equipment is needed in the method for detecting genotoxic impurities in gliclazide at the present stage, the invention provides an analysis method of the potential genotoxic impurities in gliclazide Ji Tezhong, which is used for detecting the content of the potential genotoxic impurities N-amino-3-azabicyclo [3, 0] octane in gliclazide Ji Tezhong. Firstly, preparing a sample solution by using absolute methanol as a solvent; then, the quaternary ammonium salt strong anion exchange group bonded high-purity silica gel is used as a filler to separate the N-amino-3-azabicyclo [3, 0] octane which is a potential genetic toxic impurity; and finally, adopting an electric fog type detector to analyze and detect, and realizing the control of the potential genetic toxic impurities of the glibenches Ji Tezhong.
The technical scheme of the invention is as follows:
a method of analyzing a potentially genotoxic impurity of glibenclamide Ji Tezhong, said potentially genotoxic impurity selected from the group consisting of hydrazines; firstly, preparing a sample solution by using absolute methanol as a solvent; then, the quaternary ammonium salt strong anion exchange group bonded high-purity silica gel is used as a filler to separate potential genetic toxic impurities; finally, adopting an electric fog type detector to analyze and detect;
the potentially genotoxic impurity is N-amino-3-azabicyclo [3, 0]]Octane of the structural formula
。
Preferably, the mobile phase A adopted in the separation is 0.1% aqueous solution of heptafluorobutyric acid, and the mobile phase B is methanol, and the gradient elution is carried out; the flow rate is 0.5mL/min; the column temperature is 30-40 ℃ and the sample injection volume is 20 mu L.
Preferably, the gradient elution procedure is: 0-3min, phase A: the volume ratio of the phase B is 80:20;3-10min, phase A: the volume ratio of the phase B is 80:20-20:80; 10-12min, phase A: the volume ratio of the phase B is 20:80;12-12.5min, phase A: the volume ratio of the phase B is 20:80-80:20; 12.5-20min, phase A: the volume ratio of the phase B is 80:20.
Preferably, the sampling frequency of the electric fog detector is 5Hz, the filtering constant is 3.6s, and the atomization temperature is 35-45 ℃.
Preferably, the content of the potentially genotoxic impurities in the test sample solution is calculated as peak area using an external standard method;
content (ppm) =a i /C i ×C s /A s ×10 6
Wherein: ai is the peak area of genotoxic impurities in the chromatogram of the sample solution;
as is the peak area of genotoxic impurities in the chromatogram of the reference solution;
ci is the concentration of the test sample, mg/mL;
cs is the concentration of the control, ng/mL.
The invention also aims to protect a quality control method of gliclazide bulk drug, which is measured by adopting the analysis method.
Another object of the present invention is to provide a method for quantitatively determining N-amino-3-azabicyclo [3, 0] octane, which is carried out by the above-mentioned analytical method.
The invention provides an electrospray high performance liquid chromatography analysis method which is used for separating and quantitatively analyzing N-amino-3-azabicyclo [3, 0] octane which is a potential genotoxic impurity of glibenches Ji Tezhong. The effective control of the potential genotoxic impurities of the glibenclamide Ji Tezhong is realized, the occurrence of side effects of the medication of patients is reduced, and the medication safety of the patients is ensured to a certain extent.
In order to accurately detect the content of N-amino-3-azabicyclo [3, 0] octane which is a potential genotoxic impurity of gliclazide Ji Tezhong, the invention firstly adopts anhydrous methanol as a solvent according to the solubility and structural characteristics of gliclazide, so as to realize the stability of a sample solution; secondly, selecting quaternary ammonium salt strong anion exchange groups to bond with a high-purity silica gel chromatographic column, so as to realize the selectivity of N-amino-3-azabicyclo [3, 0] octane chromatographic peaks and the separation of other chromatographic peaks; in addition, heptafluorobutyric acid is added into the mobile phase, so that column retention and symmetry of chromatographic peaks of N-amino-3-azabicyclo [3, 0] octane are realized, and finally, quantitative analysis is carried out by adopting an electric fog detector according to the structural characteristics and detection sensitivity requirements of the N-amino-3-azabicyclo [3, 0] octane. And meanwhile, the composition of the mobile phase, a gradient elution program, the temperature of the atomizer and other chromatographic conditions are screened, and finally, the optimal analysis method is determined.
The invention has the beneficial effects that:
1. the solvent anhydrous methanol used in the invention is economic and easy to obtain, has low toxicity, and does not interfere with detection and content measurement of other ultraviolet absorption impurities in gliclazide, so that the sample solution of the analysis method can be used for detection and content measurement of gliclazide-specific related substances, thereby greatly improving the working efficiency and saving the working cost.
2. The solvent anhydrous methanol used in the invention effectively avoids the degradation of gliclazide, realizes the stability of the sample solution, has the RSD of the peak area of N-amino-3-azabicyclo [3, 0] octane of 3.4% in the sample solution within 24 hours, and has higher solubility for both gliclazide and impurity N-amino-3-azabicyclo [3, 0] octane.
3. The analysis method adopts quaternary ammonium salt strong anion exchange groups to bond with a high-purity silica gel chromatographic column, effectively realizes the selectivity of N-amino-3-azabicyclo [3, 0] octane chromatographic peak and the separation of other chromatographic peaks, and has the theoretical plate number of the N-amino-3-azabicyclo [3, 0] octane peak of 17789 and the separation degree of the N-amino-3-azabicyclo [3, 0] octane peak from the adjacent peaks of 27.7.
4. According to the analysis method disclosed by the invention, 0.1% of heptafluorobutyric acid is added into a mobile phase, gradient elution is carried out, and the column retention and symmetry of the chromatographic peak of N-amino-3-azabicyclo [3, 0] octane are realized, wherein the retention time is 4.417 minutes, and the tailing factor is 0.9.
5. The analysis method provided by the invention adopts an electric fog type detector, and has the advantages of high sensitivity and good stability. The method can effectively detect the impurity N-amino-3-azabicyclo [3, 0] octane, the detection limit concentration is 2.5ng/mL, the quantitative limit concentration is 8.25ng/mL, the sensitivity requirement of a mass spectrum detector is met, meanwhile, the defect of poor signal stability of the mass spectrum detector is effectively avoided, and the RSD of the peak area of the N-amino-3-azabicyclo [3, 0] octane reference substance solution within 24 hours is 2.6%.
6. The analysis method provided by the invention has the advantages of good repeatability and high accuracy. The repetitive RSD of N-amino-3-azabicyclo [3, 0] octane is 2.1%; the average recovery was 99.1% (n=9), RSD was 2.9%.
7. The analysis method provided by the invention is used for detecting the N-amino-3-azabicyclo [3, 0] octane which is a potential genotoxic impurity, is simple and quick to operate, has low detection cost, and has stronger popularization and higher application value compared with a mass spectrum detector.
8. The method provided by the invention is verified by methodology, and the results meet the requirements, so that the method provided by the invention can be used for separating and quantitatively analyzing N-amino-3-azabicyclo [3, 0] octane which is a potential genotoxic impurity of glibenches Ji Tezhong. The invention has important significance for quality control of gliclazide bulk drug and medication safety of preparation thereof, and N-amino-3-azabicyclo [3, 0] octane is often used as a starting material and an intermediate for a synthesis process of bulk drug, so the invention also provides reference for quantitative analysis of N-amino-3-azabicyclo [3, 0] octane in other drugs.
Drawings
FIG. 1 is a blank solvent chromatogram;
FIG. 2 is a chromatogram of a control solution;
FIG. 3 is a chromatogram of a test solution;
FIG. 4 is a standard graph of N-amino-3-azabicyclo [3, 0] octane.
Detailed Description
The invention will be further illustrated with reference to specific examples, which are intended to better illustrate the content of the invention, but not to limit the invention. The experimental methods used in the examples are all conventional methods unless otherwise specified; materials, reagents and the like used are commercially available unless otherwise specified. Thus, those skilled in the art will appreciate that various modifications and adaptations can be made in light of the above teachings and yet remain within the scope of the present invention.
EXAMPLE 1 investigation of solvent
1. Preparation of test solution
Because of the specificity of gliclazide structure and physicochemical properties, we must compromise the solubility and stability of the solution, so we screen the solvent.
Taking a proper amount of the product, precisely weighing, selecting three different solvents for dissolution and dilution to prepare a solution containing about 4mg of the product in each 1mL, and shaking uniformly to serve as a test sample solution.
Solvent 1: anhydrous methanol
Solvent 2: acetonitrile
Solvent 3: acetonitrile-0.1% formic acid solution (1:1)
2. Chromatographic conditions
Instrument: a liquid chromatograph. Chromatographic column: thermo Hypersil GOLD SAX (4.6X105 mm,3 μm); the mobile phase A is 0.1% aqueous solution of heptafluorobutyric acid, the mobile phase B is methanol, and the gradient elution is carried out, wherein the flow rate is 0.5mL/min; the column temperature is 30-40 ℃, and the sample injection volume is 20 mu L; the sampling frequency of the electric fog detector is 5Hz, the filtering constant is 3.6s, and the atomization temperature is 40 ℃.
Table 1 gradient elution procedure is shown in the table below
3. Results and conclusions
TABLE 2 solvent screening results
Conclusion: as can be seen from Table 2, the sample solution was left at room temperature for 24 hours with anhydrous methanol as solvent, the RSD of the peak area of the potentially genotoxic impurity N-amino-3-azabicyclo [3, 0] octane was less than 5%, the solution stability was good, while acetonitrile and acetonitrile-0.1% formic acid solution (1:1) were used as solvents, and the solution stability was poor.
Example 2 selection of chromatographic column
1. Preparation of test solution
Taking a proper amount of the product, precisely weighing, dissolving with absolute methanol, diluting to prepare a solution containing about 4mg of the product per 1mL, and shaking uniformly to obtain a sample solution.
2. Chromatographic conditions
Three different chromatographic columns were selected and measured separately according to the chromatographic conditions defined in example 1.
Chromatographic column 1: thermo Hypersil GOLD SAX (4.6X105 mm,3 μm);
chromatographic column 2: thermo Hypersil GOLD C18 (4.6X105 mm,3 μm);
chromatographic column 3: thermo Hypersil GOLD aQ (4.6X105 mm,3 μm).
3. Results and conclusions
The Thermo Hypersil GOLD C chromatographic column is adopted, the column retention of the potential genotoxic impurity N-amino-3-azabicyclo [3, 0] octane is poor, the Thermo Hypersil GOLD aQ chromatographic column is adopted, the N-amino-3-azabicyclo [3, 0] octane peak cannot be separated from the glibenches Ji Tefeng by a baseline, and the Thermo Hypersil GOLD SAX chromatographic column is adopted, so that the retention time of the N-amino-3-azabicyclo [3, 0] octane chromatographic peak is proper, the peak type is good, and the separation degree meets the requirements with the glibenches Ji Tefeng. Thermo Hypersil GOLD SAX (4.6X105 mm,3 μm) was therefore chosen as the column.
EXAMPLE 3 investigation of the detector atomization temperature
1. Preparation of control solution
Taking a proper amount of N-amino-3-azabicyclo [3, 0] octane reference substance, precisely weighing, quantitatively dissolving with absolute methanol, diluting to prepare a solution containing about 0.04 mug in each 1mL, and shaking uniformly to serve as a reference substance solution.
2. Chromatographic conditions
The detector atomization temperature was set at 30 ℃, 35 ℃, 40 ℃, 45 ℃ according to the chromatographic conditions determined in example 1, with the other conditions unchanged.
3. Results and conclusions
Table 3 atomizer temperature investigation results
Conclusion: as is clear from Table 3, the atomization temperature was increased, the peak area of N-amino-3-azabicyclo [3, 0] octane was increased, but the change in the peak area of N-amino-3-azabicyclo [3, 0] octane was not significant at 35℃to 45℃and, in addition, considering that the sample was degraded due to the high temperature, the incomplete atomization of the sample was contaminated with the sample due to the low temperature, and finally, the stability and accuracy of the detection result were both considered, the atomizer temperature was set at 40 ℃.
EXAMPLE 4 selection of mobile phase
1. Preparation of control solution
Taking a proper amount of N-amino-3-azabicyclo [3, 0] octane reference substance, precisely weighing, quantitatively dissolving with absolute methanol, diluting to prepare a solution containing about 0.04 mug in each 1mL, and shaking uniformly to serve as a reference substance solution.
2. Chromatographic conditions
Three organic solvents with different polarities are selected as eluting phases, and are respectively measured according to set chromatographic conditions.
Eluting phase 1: acetonitrile; eluting phase 2: methanol; eluting phase 3: ethyl acetate.
3. Results and conclusions
Acetonitrile is adopted as an eluting phase, N-amino-3-azabicyclo [3, 0] octane is taken from a chromatogram of a reference substance solution, and is basically not reserved and is almost co-eluted with a solvent; ethyl acetate is adopted as eluting phase, chromatographic peak retention time is too long and tailing is serious; and methanol is used as the eluting phase, the retention time of chromatographic peaks is proper, and the repeatability of the retention time is greatly improved and the peak shape is better after 0.1% of heptafluorobutyric acid is added into the water phase. Methanol was selected as the eluting phase, and 0.1% heptafluorobutyric acid was added to the aqueous phase.
EXAMPLE 5 methodology investigation
Specificity investigation: the blank solvent does not interfere with the detection of impurity N-amino-3-azabicyclo [3, 0] octane; the blank solvent chromatogram is shown in fig. 1.
Detection limit/quantitative limit investigation: precisely measuring a proper amount of impurity N-amino-3-azabicyclo [3, 0] octane reference substance, dissolving with absolute methanol, gradually diluting, and selecting a solution with a signal to noise ratio of about 3 as a detection limit solution and a solution with a signal to noise ratio of about 10 as a quantitative limit solution.
And (3) examining the precision of the instrument: taking a reference substance solution, continuously sampling for 6 times according to the determined chromatographic conditions, and calculating RSD of the peak area of each impurity; the chromatogram of the control solution is shown in FIG. 2.
Linear investigation: taking a proper amount of impurity N-amino-3-azabicyclo [3, 0] octane reference substance, precisely weighing, quantitatively dissolving and diluting with anhydrous methanol to prepare 7 solutions with different concentrations, wherein each 1mL of solution contains about 0.009 [ mu ] g to 0.213 [ mu ] g, and performing a linear regression equation on peak areas according to the concentrations; the results are shown in FIG. 4.
Repeatability investigation: preparing 6 parts of a sample solution according to a determined method, analyzing and detecting according to a determined chromatographic condition, and calculating RSD of the impurity N-amino-3-azabicyclo [3, 0] octane content in the sample solution; the chromatogram of the sample solution is shown in FIG. 3.
The method comprises the following steps of (1) examining accuracy: a control solution was prepared according to the established method. About 40mg of the sample was precisely weighed, placed in a 10mL measuring flask, and 0.1mL (50% limit concentration), 0.2mL (100% limit concentration) and 0.3mL (150% limit concentration) of a solution containing about 2. Mu.g of N-amino-3-azabicyclo [3, 0] octane as an impurity were precisely added to each 1mL, and dissolved in anhydrous methanol and diluted to a scale to prepare 3 parts of each concentration as a sample solution. And (3) analyzing and detecting according to the determined chromatographic conditions, and calculating the recovery rate of the impurity N-amino-3-azabicyclo [3, 0] octane.
Solution stability investigation: taking a sample solution with 100% limiting concentration under the method accuracy investigation item and a reference substance solution, respectively carrying out analysis and detection at room temperature (25 ℃) for 0, 4, 8, 12, 16, 20 and 24 hours according to the determined chromatographic conditions, and calculating the RSD of the peak area of the impurity N-amino-3-azabicyclo [3, 0] octane.
Method durability inspection: and (3) taking a sample solution under the 'repeatability investigation' item, respectively changing the column temperature (+ -5 ℃) and the atomizer extraction temperature (+ -5 ℃) for analysis and detection, and keeping other chromatographic conditions unchanged. The results show that the impurity N-amino-3-azabicyclo [3, 0] octane content does not change significantly after column temperature and atomization temperature are finely adjusted.
Table 4 methodological investigation results
The above embodiments are only for illustrating the technical solution of the present invention in detail, and not for limiting the technical solution of the present invention, and those skilled in the art may modify or substitute the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all that is included in the scope of the claims of the present invention is included in the present invention.
Claims (3)
1. A method of analyzing a potentially genotoxic impurity of glibenclamide Ji Tezhong, characterized in that said potentially genotoxic impurity is selected from the group consisting of hydrazines; firstly, preparing a sample solution by using absolute methanol as a solvent; then, the quaternary ammonium salt strong anion exchange group bonded high-purity silica gel is used as a filler to separate potential genetic toxic impurities; finally, adopting an electric fog type detector to analyze and detect;
the potentially genotoxic impurity is N-amino-3-azabicyclo [3,3,0]]Octane of the structural formula
；
The mobile phase A adopted in the separation is 0.1% aqueous solution of heptafluorobutyric acid, the mobile phase B is methanol, and the gradient elution is carried out; the flow rate is 0.5mL/min; the column temperature is 30-40 ℃, and the sample injection volume is 20 mu L;
the gradient elution procedure was: 0-3min, phase A: the volume ratio of the phase B is 80:20;3-10min, phase A: the volume ratio of the phase B is 80:20-20:80; 10-12min, phase A: the volume ratio of the phase B is 20:80;12-12.5min, phase A: the volume ratio of the phase B is 20:80-80:20; 12.5-20min, phase A: the volume ratio of the phase B is 80:20;
the sampling frequency of the electric fog type detector is 5Hz, the filtering constant is 3.6s, and the atomization temperature is 40 ℃.
2. The method of claim 1, wherein the content of the potentially genotoxic impurities in the sample solution is calculated as peak area using an external standard method;
content (ppm) =ai/ci×cs/as×106
Wherein: ai is the peak area of genotoxic impurities in the chromatogram of the sample solution;
as is the peak area of genotoxic impurities in the chromatogram of the reference solution;
ci is the concentration of the test sample, mg/mL;
cs is the concentration of the control, ng/mL.
3. A quality control method of gliclazide bulk drug, characterized in that the analysis method of claim 1 or 2 is adopted for measurement.
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