CN115598267A - Analysis method of potential genotoxic impurities in gliclazide - Google Patents
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Abstract
The invention relates to the technical field of pharmaceutical analysis, relates to an analysis method of potential genotoxic impurities in gliclazide, and particularly relates to an electrospray high performance liquid chromatography analysis method of the potential genotoxic impurities, which is used for analyzing and detecting the potential genotoxic impurities N-amino-3-azabicyclo [3, 0] octane in the gliclazide. Firstly, preparing a test solution by using anhydrous methanol as a solvent; then, the quaternary ammonium salt strong anion exchange group bonded high-purity silica gel is used as a filling agent to separate N-amino-3-azabicyclo [3, 0] octane which is a potential genetic toxicity impurity; and finally, an electric spray type detector is adopted for analysis and detection, so that control on potential genotoxic impurities in the gliclazide is realized.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to an electrospray high performance liquid chromatography analysis method of potential genotoxic impurities, which is used for analyzing and detecting the potential genotoxic impurities N-amino-3-azabicyclo [3, 0] octane in gliclazide.
Background
Gliclazide (Gliclazide), chemical name 1- (3-azabicyclo [3, 0)]Octyl) -3-p-toluenesulfonylurea with molecular formula C 12 H 21 N 3 O 3 S, the structural formula is as follows:
gliclazide is a second-generation sulfonylurea oral hypoglycemic drug developed by Schvea France, has dual functions 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 a first-line oral hypoglycemic drug in China.
Regarding the synthesis of gliclazide, literature reports that cyclopentane phthalic anhydride is mostly used as a raw material, and is refluxed with hydrazine hydrate in a solvent to obtain N-amino-1, 2-cyclopentane dicarboximide which is then subjected to KBH 4 /AlCl 3 Reduction to give N-amino-3-azabicyclo [3, 0]Octane and its hydrochloride, and finally, the gliclazide is obtained through condensation reaction with the p-toluenesulfonylurea. The synthetic process route is as follows:
according to a synthetic route, N-amino-3-azabicyclo [3, 0] octane is a key intermediate for synthesizing gliclazide, an amido bond exists in a molecular structure of the gliclazide, the gliclazide is easy to degrade to generate the N-amino-3-azabicyclo [3, 0] octane, and the N-amino-3-azabicyclo [3, 0] octane is not only a process impurity of the gliclazide but also a degradation impurity. In addition, N-amino-3-azabicyclo [3, 0] octane belongs to a hydrazine compound, and is a potential genotoxic impurity, and whether the content of the impurity exceeds the limit needs to be monitored in gliclazide according to the relevant regulations of drug registration regulations and ICH M7 guidelines. The control limit of N-amino-3-azabicyclo [3, 0] octane is defined as 0.001% based on the maximum daily dose of gliclazide being 120 mg/day, calculated to have a limiting level of genotoxic impurities of less than 1.5 μ g/day.
The earlier stage research result shows that the molecular structure of the N-amino-3-azabicyclo [3, 0] octane does not contain double bonds, conjugated bonds and other functional groups with ultraviolet absorption, so that the quantitative detection of a liquid chromatogram ultraviolet detector cannot be adopted; the sensitivity of universal detectors such as a differential refraction detector, an evaporation light detector and the like is low, and the detection requirement cannot be met; although the mass spectrometer has higher sensitivity, the response signal is easily influenced by the ionization degree of the compound, the stability is poorer, the price is high, and the mass spectrometer is not easy to popularize and apply; by adopting a gas chromatography analysis method, the repeatability and the accuracy of a detection result are poor because gliclazide is degraded at high temperature to generate N-amino-3-azabicyclo [3, 0] octane.
In conclusion, in order to take structural characteristics and physicochemical properties of gliclazide and N-amino-3-azabicyclo [3, 0] octane into consideration and meet the requirement on detection sensitivity, the invention develops an electrospray high performance liquid chromatography analysis method for separating and detecting N-amino-3-azabicyclo [3, 0] octane serving as a potential genotoxic impurity in gliclazide. At present, no public report is found about 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 guarantee of preparations of the gliclazide bulk drugs.
Disclosure of Invention
Aiming at the problem that a method for detecting genotoxic impurities in gliclazide at the present stage needs high-end detection equipment, the invention provides an analysis method for the potential genotoxic impurities in the gliclazide, which is used for detecting the content of the potential genotoxic impurities N-amino-3-azabicyclo [3, 0] octane in the gliclazide. Firstly, preparing a test solution by using anhydrous methanol as a solvent; then, the quaternary ammonium salt strong anion exchange group bonded high-purity silica gel is used as a filling agent to separate N-amino-3-azabicyclo [3, 0] octane which is a potential genetic toxicity impurity; and finally, an electric spray type detector is adopted for analysis and detection, so that control on potential genotoxic impurities in the gliclazide is realized.
The technical scheme of the invention is as follows:
a method for analyzing potential genotoxic impurities in gliclazide, wherein the potential genotoxic impurities are selected from hydrazine compounds; firstly, preparing a test solution by using anhydrous methanol as a solvent; then, quaternary ammonium salt strong anion exchange group bonded high-purity silica gel is used as a filling agent to separate potential genotoxic impurities; finally, analyzing and detecting by using an electric fog type detector;
the potentially genotoxic impurity is N-amino-3-azabicyclo [3,3,0]Octane of the formula
。
Preferably, the mobile phase A adopted for separation is 0.1 percent heptafluorobutyric acid aqueous solution, the mobile phase B is methanol, and gradient elution is carried out; the flow rate is 0.5mL/min; the column temperature is 30-40 ℃, and the 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; 3-10min, phase A: the volume ratio of the phase B is 80;10-12min, phase A: the volume ratio of the phase B is 20; 12-12.5min, phase A: the volume ratio of the phase B is 20;12.5-20min, phase A: the volume ratio of the phase B is 80.
Preferably, the sampling frequency of the electric fog type detector is 5Hz, the filtering constant is 3.6s, and the atomizing temperature is 35-45 ℃.
Preferably, the content of the potential genotoxic impurities in the test solution is calculated by adopting an external standard method according to peak area;
content (ppm) = A i /C i ×C s /A s ×10 6
In the formula: ai is the peak area of genotoxic impurities in the chromatogram of the test solution;
as is the peak area of genotoxic impurities in the chromatogram of the control 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 the gliclazide bulk drug, which adopts the analysis method for determination.
Another object of the present invention is to provide a method for the quantitative determination of N-amino-3-azabicyclo [3, 0] octane 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 potential genotoxic impurity N-amino-3-azabicyclo [3, 0] octane in gliclazide. The effective control of potential genotoxic impurities in the gliclazide 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 in gliclazide, the method firstly adopts absolute methanol as a solvent according to the solubility and the structural characteristics of the gliclazide, so that the stability of a test solution is realized; secondly, a quaternary ammonium salt strong anion exchange group is selected to be bonded with a high-purity silica gel chromatographic column, so that the selectivity of an N-amino-3-azabicyclo [3, 0] octane chromatographic peak and the separation from other chromatographic peaks are realized; in addition, heptafluorobutyric acid is added into the mobile phase, the column retention and symmetry of the chromatographic peak of the N-amino-3-azabicyclo [3, 0] octane are realized, and finally, an electric spray type detector is adopted for quantitative analysis according to the structural characteristics and detection sensitivity requirements of the N-amino-3-azabicyclo [3, 0] octane. Meanwhile, the composition of the mobile phase, the 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 method is economical and easy to obtain, has low toxicity, and does not interfere the detection and content determination of other ultraviolet absorption impurities in the gliclazide, so that the test solution prepared by the analysis method can be used for the detection and content determination of gliclazide related substances, the working efficiency is improved to a great extent, and the working cost is saved.
2. The solvent used in the invention is absolute methanol, so that the degradation of gliclazide is effectively avoided, the stability of the sample solution is realized, the RSD of the peak area of N-amino-3-azabicyclo [3, 0] octane in the sample solution is 3.4% within 24 hours, and simultaneously, the solubility of gliclazide and N-amino-3-azabicyclo [3, 0] octane are higher.
3. According to the analysis method, the quaternary ammonium salt strong anion exchange group is bonded with the high-purity silica gel chromatographic column, so that the selectivity of an N-amino-3-azabicyclo [3, 0] octane chromatographic peak and the separation from other chromatographic peaks are effectively realized, the theoretical plate number of the N-amino-3-azabicyclo [3, 0] octane peak is 17789, and the separation degree from the adjacent peak is 27.7.
4. According to the analysis method, the heptafluorobutyric acid with the concentration of 0.1% is added into the mobile phase, and gradient elution is carried out, so that the column retention and symmetry of the chromatographic peak of the N-amino-3-azabicyclo [3, 0] octane are realized, the retention time is 4.417 minutes, and the tailing factor is 0.9.
5. The analysis method adopts an electric fog type detector, and has high sensitivity and good stability. The method can effectively detect N-amino-3-azabicyclo [3, 0] octane serving as an impurity, the detection limit concentration is 2.5ng/mL, the quantification limit concentration is 8.25ng/mL, the sensitivity requirement of a mass spectrum detector is met, 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 has good repeatability and high accuracy. The repetitiveness RSD of the N-amino-3-azabicyclo [3, 0] octane is 2.1 percent; the average recovery was 99.1% (n = 9) and 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 rapid to operate, is low in detection cost, and has stronger popularization and higher application value compared with a mass spectrum detector.
8. The results of the methodological verification prove that the method meets the requirements, and the method can be used for the separation and quantitative analysis of the potential genotoxicity impurity N-amino-3-azabicyclo [3, 0] octane in the gliclazide. The method has important significance on the quality control of gliclazide bulk drugs and the medication safety of preparations thereof, and simultaneously, the N-amino-3-azabicyclo [3, 0] octane is often used as a starting material and an intermediate for the synthesis process of the bulk drugs, so the method also provides reference for the quantitative analysis of the 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 sample solution;
FIG. 4 is a graph showing a standard curve of N-amino-3-azabicyclo [3, 0] octane.
Detailed Description
The invention will now be further illustrated by reference to specific examples, which are provided for the purpose of better illustrating the invention and are not to be construed as limiting the invention. The experimental methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the present invention as described above without departing from the scope of the present invention.
EXAMPLE 1 examination of solvent
1. Preparation of test solution
Due to the particularity of the structure and the physicochemical property of the gliclazide, the solubility and the stability of the solution must be considered, so that the solvent is screened.
Taking a proper amount of the product, precisely weighing, selecting three different solvents to dissolve and dilute the product to prepare a solution containing about 4mg of the product in each 1mL, and shaking up to obtain a test solution.
Solvent 1: anhydrous methanol
Solvent 2: acetonitrile (ACN)
Solvent 3: acetonitrile-0.1% formic acid solution (1
2. Chromatographic conditions
The instrument comprises: a liquid chromatograph. And (3) chromatographic column: thermo Hypersil GOLD SAX (4.6X 150mm,3 μm); the mobile phase A is a 0.1 percent heptafluorobutyric acid aqueous solution, the mobile phase B is methanol, and the gradient elution is carried out at the flow rate of 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 type detector is 5Hz, the filtration constant is 3.6s, and the atomization temperature is 40 ℃.
Table 1 gradient elution procedure is shown in the following table
3. Results and conclusions
Table 2 solvent screening results
And (4) conclusion: as can be seen from table 2, when the sample solution is left at room temperature for 24 hours using anhydrous methanol as a solvent, the RSD of the peak area of the potential genotoxic impurity N-amino-3-azabicyclo [3, 0] octane is less than 5%, and the solution stability is good, whereas when acetonitrile or acetonitrile-0.1% formic acid solution (1).
EXAMPLE 2 selection of chromatography columns
1. Preparation of test solution
Taking a proper amount of the product, precisely weighing, dissolving with anhydrous methanol, diluting to obtain a solution containing about 4mg per 1mL, and shaking to obtain a test solution.
2. Chromatographic conditions
Three different chromatographic columns were selected and tested separately according to the chromatographic conditions defined in example 1.
A chromatographic column 1: thermo Hypersil GOLD SAX (4.6X 150mm,3 μm);
and (3) chromatographic column 2: thermo Hypersil GOLD C18 (4.6X 150mm,3 μm);
and (3) chromatographic column: thermo Hypersil GOLD aQ (4.6X 150mm,3 μm).
3. Results and conclusions
The method adopts a Thermo Hypersil GOLD C18 chromatographic column, the column retention of N-amino-3-azabicyclo [3, 0] octane which is a potential genotoxic impurity is poor, adopts a Thermo Hypersil GOLD aQ chromatographic column, and the retention time of the chromatographic peak of the N-amino-3-azabicyclo [3, 0] octane and the gliclazide peak can not reach the baseline separation, but adopts a Thermo Hypersil GOLD SAX chromatographic column, and the chromatographic peak of the N-amino-3-azabicyclo [3, 0] octane has proper retention time, better peak type and meets the requirement of the separation degree of the chromatographic peak of the gliclazide. Therefore, thermo Hypersil GOLD SAX (4.6X 150mm,3 μm) was selected as the column.
EXAMPLE 3 examination of atomization temperature of Detector
1. Preparation of control solutions
Taking a proper amount of N-amino-3-azabicyclo [3, 0] octane reference substances, precisely weighing, quantitatively dissolving with anhydrous methanol, diluting to obtain a solution containing 0.04 mu g of N-amino-3-azabicyclo [3, 0] octane per 1mL, and shaking up to obtain the reference substance solution.
2. Chromatographic conditions
According to the chromatographic conditions determined in example 1, the detector atomization temperatures were set at 30 ℃, 35 ℃, 40 ℃ and 45 ℃ respectively, while the other conditions were unchanged.
3. Results and conclusions
TABLE 3 nebulizer temperature investigation results
And (4) conclusion: as can be seen from table 3, the peak area of N-amino-3-azabicyclo [3, 0] octane is increased when the atomization temperature is increased, but the peak area of N-amino-3-azabicyclo [3, 0] octane is not significantly changed when the temperature is 35 ℃ to 45 ℃, and in addition, considering that the sample may be degraded due to high temperature and the detector may be contaminated due to incomplete atomization of the sample due to low temperature, the temperature of the atomizer is set to 40 ℃ in consideration of stability and accuracy of the detection result.
EXAMPLE 4 selection of mobile phase
1. Preparation of control solutions
Taking a proper amount of N-amino-3-azabicyclo [3, 0] octane reference substances, precisely weighing, quantitatively dissolving with anhydrous methanol, diluting to obtain a solution containing 0.04 mu g of N-amino-3-azabicyclo [3, 0] octane per 1mL, and shaking up to obtain the reference substance solution.
2. Chromatographic conditions
Three organic solvents with different polarities are selected as elution phases, and the detection is respectively carried out according to set chromatographic conditions.
Elution phase 1: acetonitrile; elution phase 2: methanol; elution phase 3: and (3) ethyl acetate.
3. Results and conclusions
Acetonitrile is used as an elution phase, and N-amino-3-azabicyclo [3, 0] octane is taken from a chromatogram of a reference substance solution, basically does not remain and is almost co-eluted with a solvent; ethyl acetate is used as an elution phase, and chromatographic peak retention time is too long and serious tailing is caused; and methanol is used as an elution phase, the retention time of chromatographic peaks is appropriate, and after 0.1 percent of heptafluorobutyric acid is added into a water phase, the repeatability of the retention time is greatly improved, and the peak shape is good. Therefore, methanol was selected as the elution phase, while 0.1% heptafluorobutyric acid was added to the aqueous phase.
Example 5 methodological examination
And (3) special investigation: the blank solvent does not interfere the detection of the impurity N-amino-3-azabicyclo [3, 0] octane; the blank solvent chromatogram is shown in FIG. 1.
Detection limit/quantification limit investigation: precisely measuring a proper amount of an impurity N-amino-3-azabicyclo [3, 0] octane reference substance, dissolving by using anhydrous methanol and gradually diluting, selecting a solution with a signal-to-noise ratio of about 3 as a detection limit solution, and selecting a solution with a signal-to-noise ratio of about 10 as a quantification limit solution.
And (3) investigating instrument precision: taking a reference substance solution, carrying out continuous sample injection for 6 times according to the determined chromatographic conditions, and calculating the RSD of each impurity peak area; 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 substances, precisely weighing, quantitatively dissolving and diluting the reference substances by using anhydrous methanol to prepare 7 solutions with different concentrations of about 0.009 mu g-0.213 mu g in each 1mL, and taking a linear regression equation of concentration versus peak area; the results are shown in FIG. 4.
And (3) repeatability inspection: preparing 6 parts of a test solution according to a determined method, carrying out analysis and detection according to determined chromatographic conditions, and calculating RSD (soluble protein D) of the content of N-amino-3-azabicyclo [3, 0] octane serving as an impurity in the test solution; the chromatogram of the sample solution is shown in FIG. 3.
Method accuracy survey: control solutions were prepared according to the established method. In addition, about 40mg of the product was precisely weighed, and placed in a 10mL measuring flask, 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 per 1mL was precisely added, and dissolved and diluted to a scale with anhydrous methanol to prepare 3 parts of a test solution at each concentration. According to the analysis and detection of the determined chromatographic conditions, the recovery rate of the impurity N-amino-3-azabicyclo [3, 0] octane is calculated.
And (3) solution stability investigation: taking the sample solution and the reference solution with 100% limit concentration under the item of 'method accuracy examination', respectively analyzing and detecting at room temperature (25 ℃) for 0, 4, 8, 12, 16, 20 and 24 hours according to determined chromatographic conditions, and calculating the RSD of the peak area of the impurity N-amino-3-azabicyclo [3, 0] octane.
Method durability investigation: taking the sample solution under the 'repeatability inspection' item, respectively changing the column temperature (plus or minus 5 ℃) and the atomizer extraction temperature (plus or minus 5 ℃) for analysis and detection, and keeping other chromatographic conditions unchanged. The results show that the content of N-amino-3-azabicyclo [3, 0] octane as an impurity has no obvious change after the column temperature and the atomization temperature are adjusted finely.
TABLE 4 methodological examination results
The above embodiments are only for illustrating the technical solutions of the present invention in detail and not for limiting, and those skilled in the art can make modifications or equivalent substitutions to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and shall be covered by the claims of the present invention.
Claims (6)
1. An analysis method of potential genotoxic impurities in gliclazide, which is characterized in that the potential genotoxic impurities are selected from hydrazine compounds; firstly, preparing a test solution by using anhydrous methanol as a solvent; then, quaternary ammonium salt strong anion exchange group bonded high-purity silica gel is used as a filling agent to separate potential genotoxic impurities; finally, analyzing and detecting by using an electric fog type detector;
the potentially genotoxic impurity is N-amino-3-azabicyclo [3,3,0]Octane of the formula
。
2. The analytical method according to claim 1, wherein the separation is carried out by gradient elution using a mobile phase A of 0.1% aqueous heptafluorobutyric acid and a mobile phase B of methanol; the flow rate is 0.5mL/min; the column temperature is 30-40 ℃, and the injection volume is 20 mu L.
3. The assay method according to claim 2, wherein the gradient elution procedure is: 0-3min, phase A: the volume ratio of the phase B is 80; 3-10min, phase A: the volume ratio of the phase B is 80;10-12min, phase A: the volume ratio of the phase B is 20; 12-12.5min, phase A: the volume ratio of the phase B is 20;12.5-20min, phase A: the volume ratio of the phase B is 80.
4. The analytical method according to claim 1, wherein the sampling frequency of the electrospray detector is 5Hz, the filtration constant is 3.6s, and the atomization temperature is 35-45 ℃.
5. The assay method of claim 1, wherein the content of the potentially genotoxic impurity in the test solution is calculated as peak area using an external standard method;
content (ppm) = A i /C i ×C s /A s ×10 6
In the formula: ai is the peak area of genotoxic impurities in the chromatogram of the test solution;
as is the peak area of genotoxic impurities in the chromatogram of the control solution;
ci is the concentration of the test sample, mg/mL;
cs is the concentration of the control, ng/mL.
6. A quality control method of a gliclazide bulk drug, characterized in that the assay is performed by the assay method according to any one of claims 1 to 5.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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