CN113686981A - Method for detecting genotoxic impurities in pentoxifylline - Google Patents
Method for detecting genotoxic impurities in pentoxifylline Download PDFInfo
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- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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Abstract
The invention relates to the technical field of pharmaceutical analysis, and particularly discloses a method for detecting genotoxic impurities in pentoxifylline. The detection method comprises the following steps: preparing a test solution and a reference solution; detecting the test solution and the reference solution by adopting a liquid chromatography-mass spectrometry combined method, wherein the chromatographic conditions of the liquid chromatography are as follows: performing gradient elution by using a C18 chromatographic column and using a formic acid aqueous solution with the volume concentration of 0.1% as a mobile phase A and methanol as a mobile phase B; the mass spectrum adopts an ESI ion source and a positive ion detection mode. The detection method provided by the invention has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like, can quickly and accurately detect the genotoxic impurity D in the pentoxifylline bulk drug, and conforms to the guide principle of ICH M7.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting genotoxic impurities in pentoxifylline.
Background
The pentoxifylline is a dimethyl xanthine derivative, and can reduce blood viscosity, improve blood fluidity, promote microcirculation of ischemic tissues, and increase oxygen supply of special organs. The action mechanism is as follows: by inhibiting phosphodiesterase, the content of adenosine triphosphate in cells is increased, so that the deformability of erythrocytes is improved, fibrinogen is reduced, and the aggregation of erythrocytes and platelets is inhibited. It is mainly suitable for treating cerebral blood circulation disorder such as transient cerebral ischemia attack, cerebral apoplexy sequela, and cerebral dysfunction caused by cerebral ischemia; peripheral blood circulation disorder diseases such as thromboangiitis obliterans, etc., can protect cardiovascular and cerebrovascular, and has effects of improving heart function of patients with chronic heart failure and idiopathic dilated cardiomyopathy. Pentoxifylline is known by the English name Pentoxifylline and the chemical name 3, 7-dihydro-3, 7-dimethyl-1- (5-oxohexyl) -1H-purine-2, 6-dione, and has the following structural formula:
in the process of producing the pentoxifylline bulk drug by the prior art, a trace amount of halogenated alkane genotoxic impurity (impurity D) can be generated: 3- (3-chloropropoxy) -2-enoic acid ethyl ester, the chemical structure of which is shown below:
acceptable limits for impurity D in pentoxifylline drug substance, TTC x (1000/MDD) 1.25ppm (ng/mg), where TTC represents the toxicological threshold of interest and is 1.5 μ g/day and MDD represents the maximum daily dose and is 1200 mg/day, are as instructed by ICH M7.
The sensitivity of the conventional gas chromatography or liquid chromatography detection method cannot meet the detection requirement of the impurity D in the pentoxifylline bulk drug, and relevant literature reports of the detection method capable of meeting the requirement are found at present. Therefore, the development of a method capable of achieving the treatment of trace amounts of toxic impurities: the detection method of 3- (3-chloropropoxy) -2-ethyl enoate has important significance for the quality control of pentoxifylline.
Disclosure of Invention
In view of the above, the invention provides a method for detecting genotoxic impurities in pentoxifylline, which has excellent sensitivity, precision and linear relationship and can meet the detection requirement of 3- (3-chloropropoxy) -2-ethyl enoate in the pentoxifylline bulk drug.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a method for detecting genotoxic impurities in pentoxifylline, comprising the steps of:
step one, preparing a test solution and a reference solution;
preparing a reference substance solution: preparing a reference substance solution from 3- (3-chloropropoxy) -2-ethyl enoate by using a solvent;
preparing a test solution: taking a pentoxifylline sample, and preparing a test solution by using a solvent;
step two, detecting the test solution and the reference solution by adopting a liquid chromatography-mass spectrometry combined method, wherein the chromatographic conditions of the liquid chromatography are as follows:
performing gradient elution by using a C18 chromatographic column and using a formic acid aqueous solution with the volume concentration of 0.1% as a mobile phase A and methanol as a mobile phase B;
the mass spectrum adopts an ESI ion source and a positive ion detection mode, wherein the quantitative ions of the genotoxic impurities are as follows: the parent ion is 161m/z, the daughter ion is 85m/z, the collision voltage is 15V, the declustering voltage is 80V, and the qualitative ions of the genotoxic impurities are as follows: the parent ion is 207m/z, the daughter ion is 85m/z, the collision voltage is 25V, and the declustering voltage is 70V.
Compared with the prior art, the method for detecting genotoxic impurities in pentoxifylline provided by the invention has the following advantages:
the method for detecting genotoxic impurities in pentoxifylline provided by the application realizes quantitative and qualitative analysis of the impurities D in the pentoxifylline raw material medicine, has strong specificity and low detection limit, the detection limit of the impurities D is 1.25ng/mL, the quantification limit is 2.50ng/mL, the detection requirement of 3- (3-chloropropoxy) -2-ethyl enoate in the pentoxifylline raw material medicine is met, in addition, the linear relation is good, the repeatability is high, and the correlation coefficient in the linear range of a standard curve is more than 0.99; in addition, the detection method has the advantages of short analysis time and small sample quantity, and can greatly improve the analysis efficiency of the sample.
The method adopts a liquid chromatography-mass spectrometry combined method to detect genotoxic impurities in the pentoxifylline, has the advantages of simple and convenient method, stability, high precision, good reproducibility and the like, can quickly and accurately detect the genotoxic impurities in the pentoxifylline bulk drug, has reliable and controllable whole operation process, is suitable for practical application and popularization, and has wide application prospect.
Preferably, the gradient elution procedure in the combined liquid chromatography-mass spectrometry method is as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 2.5-3.5min, 20% → 77-83% mobile phase B, 80% → 23-17% mobile phase A;
2.5-3.5min → 8min, 77-83% mobile phase B, 23-17% mobile phase A;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
Further preferably, the procedure of gradient elution in the combined liquid chromatography-mass spectrometry is as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 3min, 20% → 80% mobile phase B, 80% → 20% mobile phase a;
3min → 8min, 80% mobile phase B, 20% mobile phase a;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
The gradient elution has obvious influence on the separation effect and the peak shape of the detection target object, so that the optimal gradient elution sequence in the application can separate the impurity D from the pentoxifylline or other interferents, the separation effect is excellent, the detection and analysis time is short, and the chromatographic peak of the impurity D can appear at 6.88 min.
Preferably, the ion source parameters of the mass spectrum are: the pressure of the ion source gas 1 is 35psi, the pressure of the ion source gas 2 is 35psi, the pressure of the gas curtain is 30psi, the temperature of the ion source is 500 ℃, the spray voltage is 5500V, the intake voltage is 10V, and the ejection voltage of the collision chamber is 10V.
Under the preferable mass spectrum analysis condition, the accuracy of the determination of the 3- (3-chloropropoxy) -2-ethyl enoate (impurity D) can be improved to the maximum extent.
Preferably, the flow rate is 0.95mL/min to 1.05mL/min, and the column temperature is 30 ℃ to 40 ℃.
More preferably, the flow rate is 1mL/min and the column temperature is 35 ℃.
The optimal flow rate and column temperature can ensure that the genotoxic impurity D can generate a peak in a short time, reduce the analysis time and have excellent separation effect by considering the chromatographic peak.
The preferred column is the type OSAKA SODA CAPCELL PAK C18.
Preferably the column has a specification of 4.6mm x 150mm x 5 μm.
Different chromatographic columns have larger difference on the retention performance of the compounds, so an OSAKA SODA CAPCELL PAK C18 column with the specification of 4.6mm multiplied by 150mm multiplied by 5 mu m is adopted in the method, the target can be quickly and effectively separated, and the peak shape is better.
Preferably, the injection volume of the liquid chromatography is 20. mu.L.
Preferably, the concentration of the control solution is 12.5 ng/mL.
Preferably, the concentration of the sample solution is 9mg/mL to 11 mg/mL.
Preferably the solvent is methanol.
Preferably, methanol is used as a solvent, so that substances except 3- (3-chloropropoxy) -2-ethyl enoate can be extracted as little as possible, 3- (3-chloropropoxy) -2-ethyl enoate in the pentoxifylline bulk drug can be extracted as much as possible, the detection of liquid chromatogram and mass spectrum is not interfered, and the detection result of the liquid chromatogram-mass spectrum combined use method provided by the invention is more accurate.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a high performance liquid chromatogram of a blank solution provided in example 1 of the present invention;
FIG. 2 is a high performance liquid chromatogram of a control solution provided in example 1 of the present invention;
FIG. 3 is a high performance liquid chromatogram of a test solution provided in example 1 of the present invention;
FIG. 4 is a high performance liquid chromatogram of the mixed solution provided in example 1 of the present invention;
FIG. 5 is a linear regression curve provided in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The embodiment provides a method for detecting genotoxic impurities in pentoxifylline, which comprises the following steps:
preparing a test solution, a reference solution, a blank solution and a mixed solution;
preparing the reference substance solution: preparing a reference substance storage solution with the concentration of 499.37ng/mL by taking a 3- (3-chloropropoxy) -2-ethyl enoate reference substance and using methanol, and then diluting the reference substance storage solution to obtain a reference substance solution with the concentration of 12.5 ng/mL;
preparing the test solution: taking 100mg of a test sample, precisely weighing, placing in a 10mL volumetric flask, adding methanol for dissolving, and diluting to a scale to obtain a test sample solution;
the blank solution is a methanol solvent;
preparing the mixed solution: 100mg of a test article is placed in a 10mL volumetric flask, 0.25mL of a control stock solution with the concentration of 499.37ng/mL is added, and methanol is added to dissolve and dilute the value scale, so that a mixed solution is obtained.
And step two, detecting the blank solution, the reference solution, the test solution and the mixed solution respectively by adopting a liquid chromatography-mass spectrometry, and recording spectrograms, wherein chromatograms of the spectrograms are respectively shown in figure 1, figure 2, figure 3 and figure 4.
The chromatographic conditions of the liquid chromatogram are as follows:
using an OSAKA SODA CAPCELL PAK C18(4.6mm × 150mm × 5 μm) chromatographic column, using a formic acid aqueous solution with a volume concentration of 0.1% as a mobile phase A and methanol as a mobile phase B, and performing gradient elution at a flow rate of 1mL/min, a column temperature of 35 ℃ and a sample injection volume of 20 μ L, wherein the procedure of the gradient elution is as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 3min, 20% → 80% mobile phase B, 80% → 20% mobile phase a;
3min → 8min, 80% mobile phase B, 20% mobile phase a;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
The conditions of the mass spectrum are as follows:
adopting ESI ion source, positive ion detecting mode, wherein the quantitative ions of genotoxic impurities are: the parent ion is 161m/z, the daughter ion is 85m/z, the collision voltage is 15V, the declustering voltage is 80V, and the qualitative ions of the genotoxic impurities are as follows: the parent ion is 207m/z, the child ion is 85m/z, the collision voltage is 25V, the declustering voltage is 70V, and the ion source parameters are as follows: the pressure of the ion source gas 1 is 35psi, the pressure of the ion source gas 2 is 35psi, the pressure of the gas curtain is 30psi, the temperature of the ion source is 500 ℃, the spray voltage is 5500V, the intake voltage is 10V, and the ejection voltage of the collision chamber is 10V.
As can be seen from FIG. 2 and FIG. 4, a chromatographic peak of the reference ethyl 3- (3-chloropropyloxy) -2-enoate (impurity D) appeared at a retention time of 6.88min, and the chromatographic peak separation degree was good.
As can be seen from the figures 1 to 4, the methanol solvent and the pentoxifylline bulk drug have no interference on the detection of the impurity D, which shows that the liquid chromatogram-mass spectrum combined usage provided by the invention has good specificity.
Example 2 detection and quantitation limits
Detection limit: the control solution with the concentration of 12.5ng/mL prepared in example 1 is diluted quantitatively by methanol step by step, and then detection is performed by adopting a liquid chromatography-mass spectrometry combined method, the specific conditions of the liquid chromatography and the mass spectrometry are as described in example 1, a spectrogram is recorded, the signal-to-noise ratio is not lower than 3:1, the detection limit is obtained, and the result is shown in Table 1.
And (4) quantitative limit: the reference solution with the concentration of 12.5ng/mL prepared in example 1 is diluted quantitatively by methanol step by step, and then the quantitative limit solution is detected by a liquid chromatography-mass spectrometry combination method, wherein the specific conditions of the liquid chromatography and the mass spectrometry are as described in example 1, a spectrogram is recorded, the quantitative limit is obtained according to the signal-to-noise ratio of not less than 10:1, and the result is shown in Table 1.
TABLE 1 detection Limit and quantitation Limit test results
6 parts of limit solution is prepared in parallel, the limit solution is detected by adopting a liquid chromatography-mass spectrometry combination method, the specific conditions of the liquid chromatography and the mass spectrometry are as described in example 1, the spectrogram is recorded, and the result is shown in table 2, thereby indicating that the limit determined by the method has excellent precision.
TABLE 2 quantitative limit repeatability test results
Example 3 Linear relationship
The control stock solution prepared in example 1 at a concentration of 499.37ng/mL was diluted with methanol to give linear solutions at concentrations of 2.50ng/mL, 4.99ng/mL, 9.99ng/mL, 12.48ng/mL, 19.97ng/mL, and 24.97ng/mL, respectively.
The linear solution prepared above was detected by a liquid chromatography-mass spectrometry combination method, the specific conditions of liquid chromatography and mass spectrometry were as described in example 1, and the spectra were recorded. A standard curve was plotted with the concentration (ng/mL) of ethyl 3- (3-chloropropyloxy) -2-enoate (impurity D) as the abscissa and the peak area as the ordinate, and a regression equation was calculated, the results are shown in Table 3, and the linear graph is shown in FIG. 5. As can be seen from the results, the concentration of impurity D in the range of 2.50ng/mL to 24.97ng/mL had a good linear relationship.
TABLE 3 impurities D Linear relationship test results
Example 4 reproducibility
Taking the same batch of samples, preparing 6 parts of test solution according to the test preparation method in the detection method of genotoxic impurities in pentoxifylline described in example 1, and detecting by adopting a liquid chromatography-mass spectrometry combined method, wherein the specific conditions of the liquid chromatography and the mass spectrometry are described in example 1, and the results are shown in Table 4. As can be seen from table 4, none of the 6 test solutions was detected, indicating that the detection method provided by the present application has good reproducibility.
TABLE 4 repeatability test results for impurity D
Example 5 accuracy
The accuracy test of the impurity D was expressed in terms of recovery (%), and limit levels of the impurities D1.00ppm, 1.25ppm, and 2.00ppm (corresponding to the impurity limits of 80%, 100%, and 160%) were taken as recovery test samples, respectively.
Preparing a reference stock solution: taking 3- (3-chloropropoxy) -2-ethyl enoate as a reference substance, and preparing a reference substance stock solution with the concentration of 499.37ng/mL by using methanol.
Preparing a reference solution: precisely measuring 0.25mL of the reference stock solution, placing in a 10mL volumetric flask, adding methanol to dilute to the scale mark, and shaking up to obtain the final product.
Preparation recovery 1.00ppm solution: taking 100.0mg of a test sample, placing the test sample in a 10mL volumetric flask, adding 0.20mL of the reference stock solution, diluting with methanol to scale marks, and shaking up to obtain the three parts in parallel.
Preparation recovery 1.25ppm solution: taking 100.0mg of a test sample, placing the test sample in a 10mL volumetric flask, adding 0.25mL of the reference stock solution, diluting with methanol to scale marks, and shaking up to obtain the three parts in parallel.
Preparation recovery 2.00ppm solution: taking 100.0mg of a test sample, placing the test sample in a 10mL volumetric flask, adding 0.40mL of the reference stock solution, diluting with methanol to scale marks, and shaking up to obtain the three parts in parallel.
The control solution and the recovery solution prepared above were subjected to detection by a liquid chromatography-mass spectrometry method, and the specific conditions of liquid chromatography and mass spectrometry were as described in example 1, and the spectra were recorded. The recovery results are shown in Table 5.
The recovery was calculated according to the following formula:
recovery (%) - (measured-original)/theoretical addition × 100%
TABLE 5 detection results of recovery of impurity D
As can be seen from Table 5, the impurity D is in the concentration range of 1.00ppm to 2.00ppm, the recovery rate is between 87.26% and 100.09%, and the RDS is less than 10%, thereby demonstrating that the method for detecting the genotoxic impurity in pentoxifylline provided by the application has good accuracy.
Example 6 precision
Taking the control solution prepared in example 1, and detecting the control solution prepared above by adopting a liquid chromatography-mass spectrometry combined method, wherein the specific conditions of the liquid chromatography and the mass spectrometry are as described in example 1, the sample injection is repeated for 6 times, 20 mu L of sample injection is performed each time, and the detection results are shown in Table 6.
TABLE 6 results of the precision test
As can be seen from Table 6, the impurity D is continuously detected for 6 times, and the peak area RSD value of the impurity D of the reference substance is 7.05% and less than 10%, thereby demonstrating that the method for detecting the genotoxic impurity in pentoxifylline provided by the application has good precision.
EXAMPLE 7 durability
The control solution prepared in example 1 at a concentration of 12.5ng/mL was assayed by a combination of liquid chromatography and mass spectrometry, the specific conditions of which were as described in example 1, and the peak areas were recorded.
Fine tuning of chromatographic conditions:
fine adjustment I and flow rate adjustment: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was assayed by a combination of liquid chromatography and mass spectrometry at a flow rate of 1.05mL/min, under the same conditions as those in example 1, and the peak area was recorded.
Fine adjustment II and flow rate adjustment: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was assayed by a combination of liquid chromatography and mass spectrometry at a flow rate of 0.95mL/min, under the same conditions as those in example 1, and the peak area was recorded.
Fine adjustment, column temperature adjustment: the control solution prepared in example 1 at a concentration of 12.5ng/mL was assayed by LC-MS at a column temperature of 40 ℃ under the same conditions as in example 1, and the peak area was recorded.
Fourthly, fine adjustment and column temperature adjustment: the control solution prepared in example 1 at a concentration of 12.5ng/mL was assayed by LC-MS at a column temperature of 30 ℃ under the same conditions as in example 1, and the peak area was recorded.
Fine adjustment five, gradient elution adjustment: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was tested by a combined liquid chromatography-mass spectrometry method, the maximum value of mobile phase B was adjusted to 83%, the other conditions of the liquid chromatography and the mass spectrometry were the same as those of example 1, and the peak areas were recorded, and the procedure of gradient elution is as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 3min, 20% → 83% mobile phase B, 80% → 17% mobile phase a;
3min → 8min, 83% mobile phase B, 17% mobile phase a;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
Fine adjustment six, gradient elution adjustment: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was tested by LC-MS with the maximum B value adjusted to 77%, and the other conditions of LC and MS were the same as those in example 1, and the peak areas were recorded, and the procedure for gradient elution is as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 3min, 20% → 77% mobile phase B, 80% → 23% mobile phase a;
3min → 8min, 77% mobile phase B, 23% mobile phase a;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
Fine adjustment seven, gradient elution adjustment: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was tested by LC-MS, the time sequence of gradient elution was adjusted, the other conditions of LC and MS were the same as those of example 1, and the peak area was recorded, and the procedure of gradient elution is as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 2.5min, 20% → 80% mobile phase B, 80% → 20% mobile phase a;
2.5min → 8min, 80% mobile phase B, 20% mobile phase a;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
Eighthly, gradient elution adjustment: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was tested by LC-MS, the time sequence of gradient elution was adjusted, the other conditions of LC and MS were the same as those of example 1, and the peak area was recorded, and the procedure of gradient elution is as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 3.5min, 20% → 80% mobile phase B, 80% → 20% mobile phase a;
3.5min → 8min, 80% mobile phase B, 20% mobile phase a;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
The detection tests are shown in Table 7, and as can be seen from Table 7, the fine-tuning chromatographic conditions have no influence on the detection of the impurity D, which indicates that the detection method for genotoxic impurities in pentoxifylline provided by the invention has good durability.
Table 7 impurity D durability test results
| Process conditions | Peak area |
| Example 1 detection conditions | 37394 |
| Fine adjustment one | 34814 |
| Fine adjustment two | 40490 |
| Fine adjustment three | 37974 |
| Fine tuning of | 37943 |
| Fine adjustment five | 38563 |
| Fine adjustment six | 35164 |
| Fine adjustment seven | 33948 |
| Fine adjustment eight | 35019 |
| Mean value of | 36812 |
| RSD(％) | 5.90 |
Example 8 intermediate precision
Taking the same batch of samples, preparing 6 parts of test solution according to the test preparation method in the detection method of genotoxic impurities in pentoxifylline described in example 1, adopting the detection date different from that of example 4 and different detectors, and detecting the prepared test solution by adopting a liquid chromatography-mass spectrometry combined method, wherein the specific conditions of the liquid chromatography and the mass spectrometry are described in example 1, and the results are shown in Table 8. The detection result shows that 6 tested sample solutions are not detected and are consistent with the repeatability result, and the intermediate precision of the detection method for genotoxic impurities in pentoxifylline provided by the application is good.
TABLE 8 results of intermediate precision test
Example 9 stability
The sample solution and the reference solution prepared in example 1 were taken and left for 0, 2, 4, 6, and 8 hours, respectively, and then the sample solution and the reference solution prepared above were tested by a liquid chromatography-mass spectrometry method, wherein the specific conditions of the liquid chromatography and the mass spectrometry were as described in example 1, 20 μ L was injected each time, a spectrum was recorded, the peak area was examined, and the RSD (%) was calculated, and the test results are shown in tables 9 and 10 below. As can be seen from tables 9-10, the test solution was left at room temperature for 8 hours without any impurity D being detected; the RSD of the peak area of the control solution is 7.03 percent (less than or equal to 10 percent) when the control solution is placed for 8 hours at room temperature, which shows that the stability of the test solution and the control solution is good.
TABLE 9 test results of solution stability of test article
TABLE 10 control solution stability test results
Example 10 sample testing
The content of impurity D in 7 samples of pentoxifylline raw materials was determined according to the method for detecting genotoxic impurities in pentoxifylline provided in example 1. The results are shown in Table 11.
TABLE 11 results of sample examination
| Batches of | Content (ng/mg) |
| 1 | Not detected out |
| 2 | Not detected out |
| 3 | Not detected out |
| 4 | Not detected out |
| 5 | Not detected out |
| 6 | Not detected out |
| 7 | Not detected out |
As can be seen from Table 7, the content of impurity D in 7 samples of the starting material of pentoxifylline was not detected and met the limit specification (. ltoreq.1.25 ppm).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for detecting genotoxic impurities in pentoxifylline is characterized in that: the detection method comprises the following steps:
step one, preparing a test solution and a reference solution;
preparing a reference substance solution: preparing a reference substance solution from 3- (3-chloropropoxy) -2-ethyl enoate by using a solvent;
preparing a test solution: taking a pentoxifylline sample, and preparing a test solution by using a solvent;
step two, detecting the test solution and the reference solution by adopting a liquid chromatography-mass spectrometry combined method, wherein the chromatographic conditions of the liquid chromatography are as follows:
performing gradient elution by using a C18 chromatographic column and using a formic acid aqueous solution with the volume concentration of 0.1% as a mobile phase A and methanol as a mobile phase B;
the mass spectrum adopts an ESI ion source and a positive ion detection mode, wherein the quantitative ions of the genotoxic impurities are as follows: the parent ion is 161m/z, the daughter ion is 85m/z, the collision voltage is 15V, the declustering voltage is 80V, and the qualitative ions of the genotoxic impurities are as follows: the parent ion is 207m/z, the daughter ion is 85m/z, the collision voltage is 25V, and the declustering voltage is 70V.
2. The method of detecting genotoxic impurities in pentoxifylline of claim 1, wherein: the procedure for the gradient elution was as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 2.5-3.5min, 20% → 77-83% mobile phase B, 80% → 23-17% mobile phase A;
2.5-3.5min → 8min, 77-83% mobile phase B, 23-17% mobile phase A;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
3. The method of detecting genotoxic impurities in pentoxifylline of claim 2, wherein: the procedure for the gradient elution was as follows:
0min → 2min, 20% mobile phase B, 80% mobile phase a;
2min → 3min, 20% → 80% mobile phase B, 80% → 20% mobile phase a;
3min → 8min, 80% mobile phase B, 20% mobile phase a;
8min → 8.1min, 80% → 20% mobile phase B, 20% → 80% mobile phase a;
8.1min → 10min, 20% mobile phase B, 80% mobile phase A.
4. The method of detecting genotoxic impurities in pentoxifylline of claim 1, wherein: the ion source parameters of the mass spectrum are as follows: the pressure of the ion source gas 1 is 35psi, the pressure of the ion source gas 2 is 35psi, the pressure of the gas curtain is 30psi, the temperature of the ion source is 500 ℃, the spray voltage is 5500V, the intake voltage is 10V, and the ejection voltage of the collision chamber is 10V.
5. The method of detecting genotoxic impurities in pentoxifylline of claim 1, wherein: the flow rate is 0.95mL/min to 1.05mL/min, and the column temperature is 30 ℃ to 40 ℃.
6. The method of detecting genotoxic impurities in pentoxifylline of claim 1, wherein: the model of the chromatographic column is OSAKA SODA CAPCELL PAK C18.
7. The method of claim 6, wherein the genotoxic impurities are detected in pentoxifylline by: the specification of the chromatographic column is 4.6mm × 150mm × 5 μm.
8. The method of detecting genotoxic impurities in pentoxifylline of claim 1, wherein: the sample injection volume of the liquid chromatography is 20 mu L.
9. The method of detecting genotoxic impurities in pentoxifylline of claim 1, wherein: the concentration of the reference substance solution is 12.5 ng/mL; and/or
The concentration of the test solution is 9 mg/mL-11 mg/mL.
10. The method for detecting genotoxic impurities in pentoxifylline according to any one of claims 1 to 9, wherein: the solvent is methanol.
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