CN113686981B - Detection method of genotoxic impurities in pentoxifylline - Google Patents
Detection method of genotoxic impurities in pentoxifylline Download PDFInfo
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Abstract
The invention relates to the technical field of medicine analysis, and particularly discloses a method for detecting genotoxic impurities in pentoxifylline. The detection method comprises the following steps: preparing a sample solution and a reference substance solution; detecting the sample solution and the reference solution by adopting a liquid chromatography-mass spectrometry method, wherein the chromatographic conditions of the liquid chromatography are as follows: adopting a C18 chromatographic column, taking formic acid water solution with the volume concentration of 0.1% as a mobile phase A, and taking methanol as a mobile phase B, and carrying out gradient elution; 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 rapidly and accurately detect the genotoxic impurity D in the pentoxifylline bulk drug, and accords with the guide principle of ICH M7.
Description
Technical Field
The invention relates to the technical field of medicine 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, thereby improving blood fluidity, promoting microcirculation of ischemic tissues and increasing oxygen supply of special organs. The action mechanism is as follows: through inhibiting phosphodiesterase, the content of adenosine triphosphate in cells is increased, so that the deformability of red blood cells is improved, fibrinogen is reduced, and aggregation of red blood cells and platelets is inhibited. It is mainly suitable for treating brain blood circulation disorder such as transient cerebral ischemia attack, cerebral apoplexy sequelae and cerebral dysfunction caused by cerebral ischemia; peripheral blood circulation disorder diseases such as thromboangiitis obliterans, etc., can protect cardiovascular and cerebrovascular diseases, and has effects of improving heart function of chronic heart failure and idiopathic dilated cardiomyopathy patients. Pentoxifyline has chemical name of 3, 7-dihydro-3, 7-dimethyl-1- (5-oxo hexyl) -1H-purine-2, 6-dione and has the following structural formula:
in the process of generating the pentoxifylline bulk drug by the prior art, trace amounts of halogenated alkane genotoxic impurities (impurities D) can be remained: 3- (3-Chloropropoxy) -2-enoic acid ethyl ester has the chemical structure shown below:
acceptable limit of impurity D in pentoxifylline drug substance = ttc× (1000/MDD) = 1.25ppm (ng/mg) according to ICH M7 guidelines, where TTC represents a toxicological concern threshold, with a value of 1.5 μg/day, MDD represents a maximum daily dose, with a value of 1200 mg/day.
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 related literature reports of the detection method capable of meeting the requirement are still available at present. Thus, a formulation was developed that enabled the achievement of a trace toxic impurity: the detection method of the 3- (3-chloropropoxy) -2-ethyl enoate has important significance for the quality control of the 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 relation, and can meet the detection requirement of 3- (3-chloropropoxy) -2-ethyl acrylate in pentoxifylline bulk drugs.
In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:
a method for detecting genotoxic impurities in pentoxifylline, said method comprising the steps of:
step one, preparing a sample solution and a reference substance solution;
preparing a reference substance solution: preparing 3- (3-chloropropoxy) -2-ethyl acrylate reference substance into reference substance solution by using a solvent;
preparing a test solution: taking a pentoxifylline sample, and preparing a sample solution by using a solvent;
detecting the sample solution and the reference solution by adopting a liquid chromatography-mass spectrometry method, wherein the chromatographic conditions of the liquid chromatography are as follows:
adopting a C18 chromatographic column, taking formic acid water solution with the volume concentration of 0.1% as a mobile phase A, and taking methanol as a mobile phase B, and carrying out gradient elution;
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 child ion is 85m/z, the collision voltage is 15V, the cluster removal voltage is 80V, and the qualitative ion of the genotoxic impurity is as follows: the parent ion is 207m/z, the child ion is 85m/z, the collision voltage is 25V, and the declustering voltage is 70V.
Compared with the prior art, the detection method of the genotoxic impurities in the pentoxifylline provided by the invention has the following advantages:
the method for detecting the genotoxic impurities in the pentoxifylline realizes quantitative and qualitative analysis of the impurities D in the pentoxifylline bulk drug, is strong in specificity and low in detection limit, the detection limit of the impurities D is 1.25ng/mL, the quantitative limit is 2.50ng/mL, the detection requirement of 3- (3-chloropropoxy) -2-ethyl enoate in the pentoxifylline bulk drug is met, 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 quick analysis time and small sample amount, and can greatly improve the sample analysis efficiency.
The method for detecting the genotoxic impurities in the pentoxifylline by adopting the liquid chromatography-mass spectrometry has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like, can rapidly 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 procedure for gradient elution in the liquid chromatography-mass spectrometry is as follows:
0min 2min,20% mobile phase B,80% mobile phase A;
2min 2.5-3.5min 20% → 77-83% mobile phase B80% → 23-17% mobile phase A;
2.5-3.5 min-8 min,77-83% mobile phase B,23-17% mobile phase A;
8 min-8.1 min,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 liquid chromatography-mass spectrometry is as follows:
0min 2min,20% mobile phase B,80% mobile phase A;
2 min-3 min,20% →80% mobile phase B,80% →20% mobile phase a;
3 min-8 min,80% mobile phase B,20% mobile phase A;
8 min-8.1 min,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 peak shape of the detection target object, so that the preferred gradient elution sequence can separate the impurity D from the pentoxifylline or other interfering substances, has excellent separation effect, and can also give consideration to short detection and analysis time and can generate a chromatographic peak of the impurity D at the position of 6.88 min.
Preferably, the ion source parameters of the mass spectrum are: the ion source gas 1 pressure was 35psi, the ion source gas 2 pressure was 35psi, the gas curtain gas pressure was 30psi, the ion source temperature was 500 ℃, the spray voltage was 5500V, the intake voltage was 10V, and the collision cell ejection voltage was 10V.
Under the preferable mass spectrometry conditions, the accuracy of the measurement of the 3- (3-chloropropoxy) -2-ethyl acrylate (impurity D) can be furthest improved.
Preferably, the flow rate is 0.95 mL/min-1.05 mL/min, and the column temperature is 30-40 ℃.
Further preferably, the flow rate is 1mL/min and the column temperature is 35 ℃.
The preferred flow rate and column temperature can enable the genotoxic impurity D to be capable of generating a peak in a short time, reducing analysis time and achieving excellent separation effect of the chromatographic peak.
The preferred type of column is OSAKA SODA CAPCELL PAK C18.
The preferred specification of the chromatographic column is 4.6mm by 150mm by 5 μm.
The difference of the retention performance of different chromatographic columns on the compound is larger, so that the OSAKA SODA CAPCELL PAK C column with the specification of 4.6mm multiplied by 150mm multiplied by 5 μm is adopted in the method, the rapid and effective separation of the target can be realized, and the peak shape is better.
The preferred sample injection volume of the liquid chromatograph is 20 μl.
The preferred concentration of the control solution is 12.5ng/mL.
Preferably, the concentration of the sample solution is 9 mg/mL-11 mg/mL.
Preferably, the solvent is methanol.
The methanol is preferably 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 chromatography and mass spectrum is not interfered, and the detection result of the liquid chromatography-mass spectrum combined method provided by the invention is more accurate.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed 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 other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
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 the control solution provided in example 1 of the present invention;
FIG. 3 is a high performance liquid chromatogram of the sample 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
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The embodiment provides a detection method of genotoxic impurities in pentoxifylline, which comprises the following steps:
step one, preparing a sample solution, a reference substance solution, a blank solution and a mixed solution;
preparing the reference substance solution: preparing a 3- (3-chloropropoxy) -2-ethyl acrylate reference substance into a reference substance stock solution with the concentration of 499.37ng/mL by using methanol, and then diluting the reference substance stock solution to obtain a reference substance solution with the concentration of 12.5ng/mL;
preparing the sample solution: taking 100mg of a test sample, precisely weighing, placing in a 10mL volumetric flask, adding methanol for dissolution, and diluting to a scale to obtain a test sample solution;
the blank solution is methanol solvent;
preparing the mixed solution: 100mg of the sample is taken and placed in a 10mL volumetric flask, 0.25mL of a control stock solution with the concentration of 499.37ng/mL is added, methanol is added for dissolution, and the scale of the dilution value is added, so that a mixed solution is obtained.
And step two, detecting the blank solution, the reference substance solution, the test substance solution and the mixed solution by adopting a liquid chromatography-mass spectrometry method, and recording spectrograms, wherein the chromatograms are respectively shown in fig. 1, fig. 2, fig. 3 and fig. 4.
The chromatographic conditions of the liquid chromatography are as follows:
OSAKA SODA CAPCELL PAK C (4.6mm.times.150mm.times.5μm) column was used, aqueous formic acid solution with volume concentration of 0.1% was used as mobile phase A, methanol was used as mobile phase B, gradient elution was performed at a flow rate of 1mL/min, column temperature of 35℃and sample injection volume of 20. Mu.L, wherein the procedure of gradient elution was as follows:
0min 2min,20% mobile phase B,80% mobile phase A;
2 min-3 min,20% →80% mobile phase B,80% →20% mobile phase a;
3 min-8 min,80% mobile phase B,20% mobile phase A;
8 min-8.1 min,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:
and adopting 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 child ion is 85m/z, the collision voltage is 15V, the cluster removal voltage is 80V, and the qualitative ion of the genotoxic impurity is as follows: the parent ion is 207m/z, the child ion is 85m/z, the collision voltage is 25V, the declustering voltage is 70V, wherein the ion source parameters are as follows: the ion source gas 1 pressure was 35psi, the ion source gas 2 pressure was 35psi, the gas curtain gas pressure was 30psi, the ion source temperature was 500 ℃, the spray voltage was 5500V, the intake voltage was 10V, and the collision cell ejection voltage was 10V.
As can be seen from FIG. 2 and FIG. 4, the chromatographic peak of the reference ethyl 3- (3-chloropropoxy) -2-enoate (impurity D) appears at a retention time of 6.88min, and the chromatographic peak separation degree is good.
From fig. 1 to fig. 4, it can be seen that the methanol solvent and the pentoxifylline bulk drug have no interference to the detection of the impurity D, which indicates that the specificity of the liquid chromatography-mass spectrometry provided by the invention is good.
Example 2 detection limit and quantitative limit
Limit of detection: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was quantitatively diluted stepwise with methanol, then detected by liquid chromatography-mass spectrometry, the specific conditions of liquid chromatography and mass spectrometry were as described in example 1, the spectra were recorded, and the detection limit was obtained at a signal-to-noise ratio of not less than 3:1, and the results are shown in Table 1.
Quantitative limit: the control solution prepared in example 1 with the concentration of 12.5ng/mL is quantitatively diluted step by adopting methanol, then the quantitative limit solution is detected by adopting a liquid chromatography-mass spectrometry, the specific conditions of liquid chromatography and mass spectrometry are as described in example 1, the spectrograms are recorded, the signal to noise ratio is not lower than 10:1, and the quantitative limit is obtained, and the result is shown in Table 1.
TABLE 1 limit of detection and quantitative limit of detection results
6 parts of quantitative limit solution are prepared in parallel, the quantitative limit solution is detected by adopting a liquid chromatography-mass spectrometry combination method, the specific conditions of liquid chromatography and mass spectrometry are as described in example 1, the spectrograms are recorded, and the results are shown in Table 2, so that the quantitative limit determined by the method has excellent precision.
TABLE 2 quantitative limited reproducibility assay results
EXAMPLE 3 Linear relationship
The control stock solution prepared in example 1 was diluted with methanol to give linear solutions of concentrations of 2.50ng/mL, 4.99ng/mL, 9.99ng/mL, 12.48ng/mL, 19.97ng/mL, and 24.97ng/mL, respectively.
The prepared linear solution is detected by adopting a liquid chromatography-mass spectrometry combined method, and the specific conditions of the liquid chromatography and mass spectrometry are as described in example 1, and a spectrogram is recorded. The concentration (ng/mL) of 3- (3-chloropropoxy) -2-ethyl enoate (impurity D) is used as an abscissa, the peak area is used as an ordinate, a standard curve is drawn, a regression equation is calculated, the result is shown in Table 3, and the linear diagram is shown in FIG. 5. As can be seen from the results, the impurity D has a good linear relationship in the concentration range of 2.50ng/mL to 24.97 ng/mL.
TABLE 3 test results of impurity D linear relationship
Example 4 repeatability
The same batch of samples was taken, 6 parts of test solutions were prepared according to the test preparation method in the method for detecting genotoxic impurities in pentoxifylline described in example 1, and detection was performed by liquid chromatography-mass spectrometry, and specific conditions of liquid chromatography and mass spectrometry were as described in example 1, and the results are shown in table 4. As can be seen from Table 4, none of the above 6 sample solutions was detected, indicating that the reproducibility of the detection method provided in the present application was good.
TABLE 4 repeatability test results of impurity D
Example 5 accuracy
Accuracy test of impurity D was expressed as recovery (%), and limit levels of impurity D1.00ppm, 1.25ppm, and 2.00ppm (corresponding to impurity limits of 80%, 100%, and 160%) were taken as recovery test samples, respectively.
Preparing a control stock solution: taking 3- (3-chloropropoxy) -2-ethyl acrylate reference substance, and preparing a reference substance stock solution with the concentration of 499.37ng/mL by using methanol.
Preparing a reference substance solution: precisely measuring 0.25mL of the reference stock solution, placing in a 10mL volumetric flask, adding methanol to dilute to scale mark, and shaking to obtain the final product.
Preparing a solution with a recovery rate of 1.00 ppm: 100.0mg of the sample is taken and placed in a 10mL volumetric flask, 0.20mL of the reference stock solution is added, methanol is used for diluting to the scale mark, and the sample is uniformly shaken, thus obtaining three parts of samples which are prepared in parallel.
Preparing a solution with a recovery rate of 1.25 ppm: 100.0mg of the sample is taken and placed in a 10mL volumetric flask, 0.25mL of the reference stock solution is added, methanol is used for diluting to the scale mark, and the sample is uniformly shaken, thus obtaining three parts of samples which are prepared in parallel.
Preparing a solution with a recovery rate of 2.00 ppm: 100.0mg of the sample is taken and placed in a 10mL volumetric flask, 0.40mL of the reference stock solution is added, methanol is used for diluting to the scale mark, and the sample is uniformly shaken, thus obtaining three parts of samples which are prepared in parallel.
The control solution and the recovery solution prepared above were detected by liquid chromatography-mass spectrometry, 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 amount)/theoretical addition amount×100%
TABLE 5 recovery rate of impurity D detection results
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%, so that the accuracy of the detection method of the genotoxic impurity in the pentoxifylline provided by the application is good.
EXAMPLE 6 precision
The control solution prepared in example 1 was taken and detected by liquid chromatography-mass spectrometry, the specific conditions of liquid chromatography and mass spectrometry were as described in example 1, and the sample was repeatedly injected 6 times, 20 μl each time, and the detection results are shown in table 6.
TABLE 6 precision test results
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%, so that the detection method of the genotoxic impurity in the pentoxifylline provided by the application is good in precision.
Example 7 durability
The control solution prepared in example 1 was tested by liquid chromatography-mass spectrometry at a concentration of 12.5ng/mL, and specific conditions of liquid chromatography and mass spectrometry were as described in example 1, and peak areas thereof were recorded.
Fine tuning of chromatographic conditions:
fine tuning one, flow rate adjustment: the control solution prepared in example 1 was tested by liquid chromatography-mass spectrometry at a concentration of 12.5ng/mL, at a flow rate of 1.05mL/min, under otherwise identical conditions to those of example 1, and the peak areas were recorded.
Fine tuning second, flow rate adjustment: the control solution prepared in example 1 was tested by liquid chromatography-mass spectrometry at a concentration of 12.5ng/mL, at a flow rate of 0.95mL/min, under otherwise identical conditions to those of example 1, and the peak areas were recorded.
Fine tuning three, column temperature adjustment: the control solution prepared in example 1 at a concentration of 12.5ng/mL was detected by liquid chromatography-mass spectrometry, the column temperature was 40℃and other conditions of liquid chromatography and mass spectrometry were the same as in example 1, and the peak areas thereof were recorded.
Fine tuning four, column temperature adjustment: the control solution prepared in example 1 at a concentration of 12.5ng/mL was detected by liquid chromatography-mass spectrometry, the column temperature was 30℃and other conditions of liquid chromatography and mass spectrometry were the same as in example 1, and the peak areas thereof were recorded.
Fine tuning and gradient elution adjustment: the control solution prepared in example 1 at a concentration of 12.5ng/mL was tested by liquid chromatography-mass spectrometry, the mobile phase B was adjusted to a maximum of 83%, other conditions of liquid chromatography and mass spectrometry were the same as in example 1, and the peak areas were recorded, and the procedure of gradient elution was as follows:
0min 2min,20% mobile phase B,80% mobile phase A;
2 min-3 min,20% →83% mobile phase B,80% →17% mobile phase a;
3 min-8 min,83% mobile phase B,17% mobile phase A;
8 min-8.1 min,80% →20% mobile phase B,20% →80% mobile phase A;
8.1min→10min,20% mobile phase B,80% mobile phase A.
Fine tuning and gradient elution adjustment: the control solution prepared in example 1 at a concentration of 12.5ng/mL was tested by liquid chromatography-mass spectrometry, the mobile phase B was adjusted to a maximum of 77%, other conditions of liquid chromatography and mass spectrometry were the same as in example 1, and the peak areas were recorded, and the procedure of gradient elution was as follows:
0min 2min,20% mobile phase B,80% mobile phase A;
2 min-3 min,20% → 77% mobile phase B,80% → 23% mobile phase a;
3 min-8 min,77% mobile phase B,23% mobile phase A;
8 min-8.1 min,80% →20% mobile phase B,20% →80% mobile phase A;
8.1min→10min,20% mobile phase B,80% mobile phase A.
Fine tuning seven, gradient elution adjustment: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was detected by liquid chromatography-mass spectrometry, the time sequence in the gradient elution was adjusted, other conditions of liquid chromatography and mass spectrometry were the same as those of example 1, and the peak areas thereof were recorded, and the procedure of the gradient elution was as follows:
0min 2min,20% mobile phase B,80% mobile phase A;
2min 2.5min 20% → 80% mobile phase B80% → 20% mobile phase A;
2.5 min-8 min,80% mobile phase B,20% mobile phase A;
8 min-8.1 min,80% →20% mobile phase B,20% →80% mobile phase A;
8.1min→10min,20% mobile phase B,80% mobile phase A.
Fine tuning eight, gradient elution adjustment: the control solution prepared in example 1 and having a concentration of 12.5ng/mL was detected by liquid chromatography-mass spectrometry, the time sequence in the gradient elution was adjusted, other conditions of liquid chromatography and mass spectrometry were the same as those of example 1, and the peak areas thereof were recorded, and the procedure of the gradient elution was 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;
3.5 min-8 min,80% mobile phase B,20% mobile phase A;
8 min-8.1 min,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, so that the method for detecting the genotoxic impurity in the pentoxifylline provided by the invention has good durability.
TABLE 7 results of impurity D durability test
| Method conditions | Peak area |
| Example 1 detection conditions | 37394 |
| Fine tuning one | 34814 |
| Fine tuning II | 40490 |
| Fine tuning three | 37974 |
| Fine tuning four | 37943 |
| Fine tuning five | 38563 |
| Fine tuning six | 35164 |
| Fine tuning seven | 33948 |
| Fine tuning eight | 35019 |
| Average value of | 36812 |
| RSD(％) | 5.90 |
EXAMPLE 8 intermediate precision
The same batch of samples was taken, 6 sample solutions were prepared according to the method for preparing samples in the method for detecting genotoxic impurities in pentoxifylline described in example 1, the sample solutions prepared above were detected by using different detection dates and different detectors from those of example 4, and the specific conditions of liquid chromatography and mass spectrometry were as described in example 1, and the results are shown in table 8. The detection result shows that none of the 6 measured sample solutions is detected, and the measured sample solutions are consistent with the repeatability result, so that the intermediate precision of the detection method for the genotoxic impurities in the 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 respectively left for 0, 2, 4, 6 and 8 hours, then the sample solution and the reference solution prepared above were detected by a liquid chromatography-mass spectrometry method, the specific conditions of the liquid chromatography and mass spectrometry were as described in example 1, 20 μl of each sample was introduced, the spectrogram was recorded, the peak area was examined, the RSD (%) was calculated, and the test results were as shown in tables 9 and 10 below. As can be seen from tables 9 to 10, the sample solution was left for 8 hours at room temperature, and no impurity D was detected; the reference substance solution is placed for 8 hours at room temperature, and the RSD of the peak area is 7.03 percent (less than or equal to 10 percent), which shows that the stability of the test substance solution and the reference substance solution is good.
TABLE 9 test results of stability of test solutions
Table 10 results of stability test of control solution
Example 10 sample detection
According to the method for detecting genotoxic impurities in pentoxifylline provided in example 1, the impurity D content in 7 batches of pentoxifylline raw material samples was detected. The results are shown in Table 11.
TABLE 11 sample detection results
| Batch of | Content (ng/mg) |
| 1 | Not detected |
| 2 | Not detected |
| 3 | Not detected |
| 4 | Not detected |
| 5 | Not detected |
| 6 | Not detected |
| 7 | Not detected |
As can be seen from Table 7, the impurity D content in the 7 batches of pentoxifylline feed samples was undetected, and met the limit specification (. Ltoreq.1.25 ppm).
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (9)
1. A method for detecting genotoxic impurities in pentoxifylline is characterized by comprising the following steps of: the detection method comprises the following steps:
step one, preparing a sample solution and a reference substance solution;
preparing a reference substance solution: preparing 3- (3-chloropropoxy) -2-ethyl acrylate reference substance into reference substance solution by using a solvent;
preparing a test solution: taking a pentoxifylline sample, and preparing a sample solution by using a solvent;
detecting the sample solution and the reference solution by adopting a liquid chromatography-mass spectrometry method, wherein the chromatographic conditions of the liquid chromatography are as follows:
adopting a C18 chromatographic column, taking a formic acid aqueous solution with the volume concentration of 0.1% as a mobile phase A, taking methanol as a mobile phase B, and carrying out gradient elution, wherein the gradient elution comprises the following procedures:
0min 2min,20% mobile phase B,80% mobile phase A,
2min 2.5-3.5min 20% → 77-83% mobile phase B80% → 23-17% mobile phase A,
2.5-3.5 min-8 min,77-83% mobile phase B,23-17% mobile phase A,
8 min-8.1 min,80% →20% mobile phase B,20% →80% mobile phase A,
8.1 min-10 min,20% mobile phase B,80% mobile phase A;
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 child ion is 85m/z, the collision voltage is 15V, the cluster removal voltage is 80V, and the qualitative ion of the genotoxic impurity is as follows: the parent ion is 207m/z, the child ion is 85m/z, the collision voltage is 25V, and the declustering voltage is 70V.
2. The method for detecting genotoxic impurities in pentoxifylline according to claim 1, wherein: the procedure for the gradient elution was as follows:
0min 2min,20% mobile phase B,80% mobile phase A;
2 min-3 min,20% →80% mobile phase B,80% →20% mobile phase a;
3 min-8 min,80% mobile phase B,20% mobile phase A;
8 min-8.1 min,80% →20% mobile phase B,20% →80% mobile phase A;
8.1min→10min,20% mobile phase B,80% mobile phase A.
3. The method for detecting genotoxic impurities in pentoxifylline according to claim 1, wherein: the ion source parameters of the mass spectrum are as follows: the ion source gas 1 pressure was 35psi, the ion source gas 2 pressure was 35psi, the gas curtain gas pressure was 30psi, the ion source temperature was 500 ℃, the spray voltage was 5500V, the intake voltage was 10V, and the collision cell ejection voltage was 10V.
4. The method for detecting genotoxic impurities in pentoxifylline according to claim 1, wherein: the flow rate is 0.95 mL/min-1.05 mL/min, and the column temperature is 30-40 ℃.
5. The method for detecting genotoxic impurities in pentoxifylline according to claim 1, wherein: the model of the chromatographic column is OSAKASODA CAPCELL PAK C.
6. The method for detecting genotoxic impurities in pentoxifylline according to claim 5, wherein: the specification of the chromatographic column is 4.6mm multiplied by 150mm multiplied by 5 mu m.
7. The method for detecting genotoxic impurities in pentoxifylline according to claim 1, wherein: the sample injection volume of the liquid chromatograph is 20 mu L.
8. The method for detecting genotoxic impurities in pentoxifylline according to claim 1, wherein: the concentration of the reference substance solution is 12.5ng/mL; and/or
The concentration of the sample solution is 9 mg/mL-11 mg/mL.
9. The method for detecting genotoxic impurities in pentoxifylline according to any one of claims 1 to 8, characterized by comprising the steps of: the solvent is methanol.
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| CN106309362A (en) * | 2016-11-08 | 2017-01-11 | 石药集团欧意药业有限公司 | Pentoxifylline injection composition and preparation method thereof |
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| Title |
|---|
| Mohamed A. Korany 等.A validated stability indicating DAD–HPLC method for determination of pentoxifylline in presence of its pharmacopeial related substances.Bulletin of Faculty of Pharmacy, Cairo University.2013,第51卷(第2期),211-219. * |
| 杜碧莹 ; .高效液相色谱法测定注射用己酮可可碱中有关物质.中国药业.2015,(第21期),123-125. * |
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