CN113267583A - HPLC-based nifedipine related substance analysis method - Google Patents
HPLC-based nifedipine related substance analysis method Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- 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
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Abstract
The invention provides an HPLC-based nifedipine related substance analysis method, which is used for separating and detecting related substances in nifedipine by an HPLC method, wherein the related substances comprise an impurity I and an impurity II, and the method mainly comprises the following steps: preparing a test solution: grinding a test sample by adding trichloromethane, transferring the ground sample into a volumetric flask by using methanol, and diluting to obtain a test sample solution; preparing a reference stock solution; preparing a control solution; a system utility solution; detecting the test solution and the reference solution by high performance liquid chromatography; according to the HPLC-based nifedipine related substance analysis method provided by the invention, the configuration method of the test solution is specifically limited, so that the operation error rate of an experimenter can be obviously reduced, and the detection efficiency and the correctness of a detection result are improved.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly relates to a nifedipine related substance analysis method based on HPLC.
Background
When the related substances of actually produced medicines are detected by adopting the medicine standard of the HPLC method, if the durability of the detection method is not good, and when an abnormal peak occurs, the problem of testing is not eliminated, so that the problem often comes up to the production of the batch of medicines, serious quality detection misjudgment is generated, not only is multiple quality rechecks required, but also the batch which meets the quality requirement can not be delivered from a factory. Nifedipine is a first-generation calcium antagonist, is a medicine for preventing hypertension and angina pectoris, is one of the popular medicines in the middle of the 80 th 20 th century, has quick response and high peak-to-valley ratio, and the existing HPLC analysis method for nifedipine related substances still has an optimization space in durability.
Disclosure of Invention
In order to solve the technical problems, the invention provides a nifedipine related substance analysis method based on HPLC.
The invention provides an HPLC-based nifedipine related substance analysis method, wherein related substances comprise an impurity I and an impurity II, the impurity I is nitropyridine, and the impurity II is nitrosopyridine, the method mainly comprises the following steps:
preparing a test solution: grinding a test sample by adding trichloromethane, transferring the ground sample into a volumetric flask by using methanol, diluting the ground sample to obtain a test sample solution, and storing the test sample solution in a dark place;
preparation of control stock solutions: taking an impurity I reference substance and an impurity II reference substance, respectively adding methanol to dissolve and quantitatively diluting to prepare a mixed solution;
preparation of control solutions: taking a test solution and a reference stock solution, and quantitatively diluting with a mobile phase respectively;
system applicability solution: dissolving nifedipine, an impurity I reference substance and an impurity II reference substance in methanol and diluting to obtain system applicability solutions, wherein each 1ml of the system applicability solution contains 1mg, 10 mu g and 10 mu g of nifedipine, the impurity I reference substance and the impurity II reference substance respectively;
detecting the test solution and the control solution by a high performance liquid chromatograph;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-water (60:40) is used as a mobile phase, the detection wavelength is 235nm, and the sample injection volume is 20 mu l;
system applicability requirements: in a system applicability solution chromatogram, the separation degrees between an impurity I peak, an impurity II peak and a nifedipine peak meet the requirement;
the determination method comprises the following steps: respectively injecting the test solution and the control solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 2 times;
limitation: if a chromatographic peak with the retention time consistent with that of the impurity I peak and that of the impurity II peak exists in a chromatogram of a test solution, the peak areas of the chromatographic peaks are not more than 0.1% by calculation according to an external standard method, the peak areas of other single impurities are not more than 0.2% of the peak area of nifedipine in a control solution, and the total amount of the impurities is not more than 0.5%.
Wherein, the nitropyridine is dimethyl 2, 6-dimethyl-4- (2-nitrophenyl) -3, 5-pyridinedicarboxylate, and the nitrosopyridine is dimethyl 2, 6-dimethyl-4- (2-nitrosophenyl) -3, 5-pyridinedicarboxylate.
Further, the specific method for preparing the test solution comprises the following steps: grinding the sample with chloroform at 20-30 deg.C for 3-5 min at 60-150 r/min, transferring into volumetric flask with methanol, diluting to obtain sample solution, and storing in dark place.
Further, the steps for preparing the control solution were as follows: taking the sample solution and the reference stock solution, and quantitatively diluting with a mobile phase respectively to prepare a mixed solution containing 2 mu g of nifedipine, 1 mu g of impurity I and 1 mu g of impurity II in each 1 ml.
Further, the grinding time was 3min, the grinding speed was 150r/min, and the grinding temperature was 30 ℃.
Further, the grinding time was 4min, the grinding speed was 100r/min, and the grinding temperature was 25 ℃.
Further, the grinding time was 5min, the grinding speed was 60r/min, and the grinding temperature was 20 ℃.
Further, the test solution contained 1mg of the test substance per 1ml of methanol.
The invention also provides an HPLC-based nifedipine related substance analysis method, wherein related substances comprise an impurity I and an impurity II, the impurity I is nitropyridine, and the impurity II is nitrosopyridine, and the method mainly comprises the following steps:
preparing a test solution: grinding a test sample by adding trichloromethane, transferring the ground sample into a volumetric flask by using methanol, diluting the ground sample to obtain a test sample solution, and storing the test sample solution in a dark place;
preparing an alternative test solution: grinding the sample with chloroform at 20-30 deg.C for 3-5 min at 60-150 r/min, transferring into volumetric flask with methanol, and diluting to obtain sample solution;
preparation of control stock solutions: taking an impurity I reference substance and an impurity II reference substance, respectively adding methanol to dissolve and quantitatively diluting to prepare a mixed solution;
preparation of control solutions: taking a test solution and a reference stock solution, and quantitatively diluting by using a mobile phase respectively to prepare a mixed solution containing 2 mu g of nifedipine, 1 mu g of impurity I and 1 mu g of impurity II in each 1 ml;
system applicability solution: dissolving nifedipine, an impurity I reference substance and an impurity II reference substance in methanol and diluting to obtain system applicability solutions, wherein each 1ml of the system applicability solution contains 1mg, 10 mu g and 10 mu g of nifedipine, the impurity I reference substance and the impurity II reference substance respectively;
detecting the test solution and the control solution by a high performance liquid chromatograph;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-water (60:40) is used as a mobile phase, the detection wavelength is 235nm, and the sample injection volume is 20 mu l;
the determination method comprises the following steps: respectively injecting the test solution and the control solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 2 times;
limitation: if a chromatographic peak with the retention time consistent with that of the impurity I peak and that of the impurity II peak exists in a chromatogram of a test solution, the chromatographic peak is not more than 0.1% when the chromatographic peak is calculated by an external standard method according to the peak area, the peak area of other single impurities is not more than the peak area of nifedipine in a control solution, and the total amount of the impurities is not more than 0.5%; and when the chromatographic peak with the retention time of the impurity I peak is calculated by the peak area according to an external standard method and exceeds 0.1 percent, injecting the alternative sample solution into the liquid chromatograph, recording the chromatogram until the retention time of the main component peak is 2 times, and carrying out limit calculation again.
Further, in the preparation of the candidate test solution, the grinding time is 3min, the grinding speed is 150r/min, and the grinding temperature is 30 ℃.
Further, in the preparation of the candidate sample solution, the grinding time is 4min, the grinding speed is 100r/min, and the grinding temperature is 25 ℃.
Further, in the preparation of the candidate test solution, the grinding time is 5min, the grinding speed is 60r/min, and the grinding temperature is 20 ℃.
The related substance analysis method provided by the invention has the following technical effects: the HPLC-based nifedipine related substance analysis method provided by the invention has the advantages that the detection efficiency and the correctness of the detection result are improved by specifically limiting the preparation method of the test solution, and the durability is good.
Drawings
FIG. 1 is a chromatogram obtained from analysis of nifedipine-related substances by the method of example 1;
FIG. 2 is a schematic diagram showing the comparison of chromatograms obtained by analyzing 9 batches of nifedipine related substances by a high performance liquid chromatography;
fig. 3 is a chromatogram comparison schematic diagram consisting of three chromatograms obtained by analyzing nifedipine-related substances by using a high performance liquid chromatography, wherein the three chromatograms are respectively a chromatogram obtained after a control solution is injected into a chromatograph, a chromatogram obtained after a normal test sample solution is injected into the chromatograph, and a chromatogram obtained after a test sample solution prepared by misoperation is injected into the chromatograph;
FIG. 4 is a chromatogram obtained by detecting sorafenib tosylate tablets, glatiramer hydrochloride capsules, omeprazole enteric-coated tablets, galanthamine tablets and celecoxib capsules by a high performance liquid chromatography.
Detailed Description
Example 1
The embodiment provides a nifedipine related substance analysis method based on HPLC, the related substance comprises an impurity I and an impurity II, the impurity I is nitropyridine, the impurity II is nitrosopyridine, the method mainly comprises the following steps:
preparing a test solution: grinding 100mg of a sample by adding 2ml of trichloromethane under the condition of 30 ℃, wherein the grinding time is 3min, the grinding speed is 150r/min, then transferring the sample into a 100ml volumetric flask by using 2ml of methanol, and obtaining a sample solution by constant volume through the methanol;
preparation of control stock solutions: taking a nitropyridine reference substance and a nitrosopyridine reference substance, respectively adding methanol to dissolve and quantitatively diluting to prepare a mixed solution containing 10 mu g of the nitropyridine reference substance and the nitrosopyridine reference substance per 1ml of methanol;
preparation of control solutions: taking a test solution and a reference stock solution, and quantitatively diluting by using a mobile phase respectively to prepare a mixed solution containing 2 mu g of nifedipine, 1 mu g of impurity I and 1 mu g of impurity II in each 1 ml;
system applicability solution: dissolving nifedipine, an impurity I reference substance and an impurity II reference substance in methanol and diluting to obtain system applicability solutions, wherein each 1ml of the system applicability solution contains 1mg, 10 mu g and 10 mu g of nifedipine, the impurity I reference substance and the impurity II reference substance respectively;
detecting the test solution and the control solution by a high performance liquid chromatograph;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-water (60:40) is used as a mobile phase, the detection wavelength is 235nm, and the sample injection volume is 20 mu l;
system applicability requirements: in a system applicability solution chromatogram, the separation degrees between an impurity I peak, an impurity II peak and a nifedipine peak meet the requirement;
the determination method comprises the following steps: respectively injecting the test solution and the control solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 2 times;
limitation: if a chromatographic peak with the retention time consistent with that of the impurity I peak and that of the impurity II peak exists in a chromatogram of a test solution, the chromatographic peak is not more than 0.1% when the chromatographic peak is calculated by an external standard method according to the peak area, the peak area of other single impurities is not more than the peak area of nifedipine in a control solution, and the total amount of the impurities is not more than 0.5%;
the chromatogram obtained from the analytical method of example 1 is shown in FIG. 1.
Example 2
The present embodiment provides an HPLC-based analysis method for nifedipine-related substances, which is different from embodiment 1 in that the preparation method of the sample solution is different, and the method for preparing the sample solution in this embodiment is as follows: grinding 100mg of sample with 2ml of chloroform at 25 ℃ for 4min at a grinding speed of 100r/min, transferring the sample into a 100ml volumetric flask with methanol, diluting to obtain a sample solution, and storing in dark place.
Example 3
The present embodiment provides an HPLC-based analysis method for nifedipine-related substances, which is different from embodiment 1 in that the preparation method of the sample solution is different, and the method for preparing the sample solution in this embodiment is as follows: grinding 100mg of sample with 2ml of chloroform at 20 ℃ for 5min at a speed of 60r/min, transferring the sample into a 100ml volumetric flask with methanol, diluting to obtain a sample solution, and storing in dark place.
Example 4
The embodiment provides an HPLC-based nifedipine related substance analysis method, which further comprises the following steps:
the method mainly comprises the following steps of:
preparing a test solution: grinding a test sample by adding trichloromethane, transferring the ground sample into a volumetric flask by using methanol, diluting the ground sample to obtain a test sample solution, and storing the test sample solution in a dark place;
preparing an alternative test solution: grinding 100mg of a test sample by adding trichloromethane for 3min at a grinding speed of 150r/min and a grinding temperature of 30 ℃, transferring the test sample into a 100ml volumetric flask by using methanol, and diluting to obtain a test sample solution.
Preparation of control stock solutions: taking an impurity I reference substance and an impurity II reference substance, respectively adding methanol to dissolve and quantitatively diluting to prepare a mixed solution;
preparation of control solutions: taking a test solution and a reference stock solution, and quantitatively diluting by using a mobile phase respectively to prepare a mixed solution containing 2 mu g of nifedipine, 1 mu g of impurity I and 1 mu g of impurity II in each 1 ml;
system applicability solution: dissolving nifedipine, an impurity I reference substance and an impurity II reference substance in methanol and diluting to obtain system applicability solutions, wherein each 1ml of the system applicability solution contains 1mg, 10 mu g and 10 mu g of nifedipine, the impurity I reference substance and the impurity II reference substance respectively;
detecting the test solution and the control solution by a high performance liquid chromatograph;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-water (60:40) is used as a mobile phase, the detection wavelength is 235nm, and the sample injection volume is 20 mu l;
the determination method comprises the following steps: respectively injecting the test solution and the control solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 2 times;
limitation: if a chromatographic peak with the retention time consistent with that of the impurity I peak and that of the impurity II peak exists in a chromatogram of a test solution, the chromatographic peak is not more than 0.1% when the chromatographic peak is calculated by an external standard method according to the peak area, the peak area of other single impurities is not more than the peak area of nifedipine in a control solution, and the total amount of the impurities is not more than 0.5%; and when the chromatographic peak with the retention time of the impurity I peak is calculated by the peak area according to an external standard method and exceeds 0.1 percent, injecting the alternative sample solution into the liquid chromatograph, recording the chromatogram until the retention time of the main component peak is 2 times, and carrying out limit calculation again.
Test example 1
Test sample
The method is characterized in that 9 batches of products are detected, the same batch of nifedipine raw materials is used, the batch number is 2008014, related substances of the batch of raw materials are normal (< 0.05%), and the sample batch numbers of the nifedipine sustained release tablets are respectively as follows: 20120041, 20120061, 20120071, 20120081, 20120091, 20120101, 20120111, 20120121, 20120131.
The detection method comprises the following steps:
the related substances comprise impurities I and II; the impurity I is nitropyridine, and the impurity II is nitrosopyridine;
the method mainly comprises the following steps:
preparing a test solution: grinding a test sample by adding trichloromethane, transferring the ground sample into a volumetric flask by using methanol, diluting the ground sample to obtain a test sample solution, and storing the test sample solution in a dark place;
preparation of control stock solutions: taking a nitropyridine reference substance and a nitrosopyridine reference substance, respectively adding methanol to dissolve and quantitatively diluting to prepare a mixed solution containing 10 mu g of the nitropyridine reference substance and the nitrosopyridine reference substance per 1ml of methanol;
preparation of control solutions: taking a test solution and a reference stock solution, and quantitatively diluting by using a mobile phase respectively to prepare a mixed solution containing 2 mu g of nifedipine, 1 mu g of impurity I and 1 mu g of impurity II in each 1 ml;
system applicability solution: dissolving nifedipine, an impurity I reference substance and an impurity II reference substance in methanol and diluting to obtain system applicability solutions, wherein each 1ml of the system applicability solution contains 1mg, 10 mu g and 10 mu g of nifedipine, the impurity I reference substance and the impurity II reference substance respectively;
checking equipment and instruments used in the experiment before the experiment, checking all reference substances when the equipment and instruments are normal in operation and in the valid period, checking instrument methods and experimental methods when the samples are correct and in the valid period, checking the experimental site, and checking that the volumetric flask and the pipette are correctly used and the solution of the test sample has no abnormal condition when the samples are consistent with the file requirements;
detecting the test solution and the control solution by a high performance liquid chromatograph;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-water (60:40) is used as a mobile phase, the detection wavelength is 235nm, and the sample injection volume is 20 mu l;
the determination method comprises the following steps: respectively injecting the test solution and the control solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 2 times;
limitation: if a chromatographic peak with the retention time consistent with that of the impurity I peak and that of the impurity II peak exists in a chromatogram of a test solution, the peak areas of the chromatographic peaks are not more than 0.1% by calculation according to an external standard method, the peak areas of other single impurities are not more than the peak area of nifedipine in a control solution, and the total amount of the impurities is not more than 0.5%.
The reference solution is prepared by diluting the test solution I by 50 times and adding a reference stock solution, wherein the peak area of the impurity I in 20120101 test solutions I is about 250 times of the peak area of the impurity I in a normal sample, but the peak area of the reference II is 20120101.
As shown in fig. 2, when nifedipine related substance analysis is performed by using a high performance liquid phase, only 1 of 9 batches of samples are abnormal and unqualified, and the possibility that impurities exceed the standard due to unqualified raw material medicines is eliminated, as shown in fig. 3, an unknown peak appears between two impurity peaks, which is primarily suspected to be an abnormal peak caused by pollution or HPLC failure in the preparation process of 20120101 batches of test solution, so that a hypothesis test is further proved;
hypothesis experiment
Assuming experiment 1:
and (3) suspected of 20120101 contamination of the liquid phase small bottle of the test solution I or the contamination in the liquid phase sample injection process to cause the result of impurities to exceed the standard, and re-detecting the test solution I in the original liquid phase small bottle solution and the original volumetric flask. The results are shown in Table 1.
Table 1. test results of the solutions of the sample i in the original liquid phase vial and the original volumetric flask.
| Batch number 20120101 | Peak area of impurity I |
| First time experimental data | 1392361 |
| Original liquid phase small bottle | 1393031 |
| Original volumetric flask solution | 1420072 |
And (4) conclusion: through comparison of the experimental data, the three data are basically the same, so that the pollution reasons in the liquid phase vial and the sample injection process are eliminated.
Examining TM264-07 method for measuring the content of nifedipine sustained release tablets (I) 10mg, the inventors suspect that contamination of the test solution I in the original volumetric flask caused by contamination of the glass mortar or volumetric flask resulted in abnormal results because 9 samples of this test were ground and pulverized with the same agate in a glass mortar, 9 glass mortars and 9 volumetric flasks were used for preparing the test solution I, and the agate mortar was wiped with dust-free paper between different batches, and since the content of the foreign matter I was normal in both the front and rear lots of 20120101, contamination of the agate mortar was excluded, thus proving that no problem was found in 20120101 samples.
Assuming experiment 2: supposing that experiment 1 suspects that the result is abnormal due to the pollution of a glass mortar or a volumetric flask in the process of preparing the test solution from the fine powder, 20120101 batches of fine powder are stored in dark, and a laboratory technician discards the powder after the preparation of the test solution is finished, so that a 20120101 sample needs to be taken again for detection.
As a result: the peak area of the impurity I is 11062;
and (4) conclusion: the experimental result meets the acceptable standard, so the abnormal result caused by the pollution of a glass mortar or a volumetric flask in the preparation process of the solution of the test article I is suspected;
in order to further prove that the abnormal peak is not a peak generated by pollution, hypothesis experiments are carried out to prove that if the pollution is only possible to be the pollution of a glass mortar or a volumetric flask, the glass mortar is only used for detecting nifedipine through investigation, so that the possibility of the pollution is not existed, and a 100ml volumetric flask laboratory only carries out the detection of the sorafenib tosylate, the alogliptin hydrochloride capsule, the omeprazole enteric-coated tablet, the galantamine tablet and the celecoxib capsule, and if the pollution is possible to be the products.
Hypothesis experiments: weighing 1mg of each product reference substance, preparing the product reference substance into a 100ml volumetric flask, positioning a mixed solution of nifedipine impurities I and II, and performing a liquid phase experiment by using a detection method of nifedipine related substances.
As a result: only the omeprazole and the sorafenib tablets generate peaks, so the possibility of pollution of the galantamine tablets, the glatiramer hydrochloride capsules and the celecoxib capsules is eliminated, and the possibility of pollution of the sorafenib is eliminated because the sorafenib peak generating positions are different from abnormal peaks, the positions of the peaks of the omeprazole are similar to the abnormal peaks but have different peak types (as shown in figure 4), and the latest detection product in a laboratory is checked, and the omeprazole is not detected in nearly two months, so the possibility of pollution of the omeprazole is eliminated.
Historical review comparison
OOS and OOT related to nifedipine related substance detection are investigated, abnormal conditions of inspection results which are obtained after four times of inspection are found, namely OOS1301001, OOT1501001, OOS1508006 and OOT1605002, and specific investigation process is checked to find that: when OOS1301001 nifedipine (superfine powder) is used for carrying out related substance detection, the detection result of nifedipine impurity II exceeds an acceptable standard, and the basic reason is that the detection result of nifedipine impurity II exceeds the standard due to insufficient light-shielding facilities.
When OOT1501001 nifedipine sustained-release tablets (I) are used for detecting 10mg related substances, the results of 4 batches of nifedipine impurities II meet the acceptable standard, but the results are higher than those of other batches detected on the same day, and the root cause is that the small liquid-phase bottles of the 4 batches of samples are placed for 20min under natural light in the experimental process, so that the solution is decomposed by light to generate the nifedipine impurities II.
When OOS1508006 audits data, the related substances are found to be calculated incorrectly, and after the correct calculation, the detection result of nifedipine impurities II in the detection result of nifedipine (ultrafine powder) exceeds the acceptable standard, and the fundamental reason is that the samples are not completely shielded from light, so that the nifedipine impurities II are generated by light decomposition, and the detection result exceeds the standard.
According to the abnormal condition investigation process, OOS1301001, OOT1501001 and OOS1508006 are all caused by unreasonable light shielding to decompose nifedipine impurities II in the sample, and OOS has no similarity at this time, and OOT1605002 is similar to the abnormal condition at this time.
When OOT1605002 nifedipine sustained-release tablets (I) are released at 10mg, 16030231, 16040281, 16040291, 16040301 and 16040311 batches of related substance data are found to meet the regulations, but the content of unknown impurities is larger than the previous data, the basic reason is that chloroform is a reason for generating the abnormal area of the unknown impurity peak, but the area is not enough to reach the abnormal peak area, and the existence of unknown factors is also a reason for causing the abnormal unknown impurity peak of nifedipine related substances.
Examining the specific investigation process of OOT1605002, finding that the peak-off time of unknown impurities is between impurities I and II, and the area is 100000-550000, and through OOT1605002 hypothesis experiments, it can be known that if trichloromethane is not added into a solvent, the peak of the unknown impurities does not appear, but an unknown peak with an uncertain peak area appears when 2ml of trichloromethane is added, and the sample is ground by trichloromethane, so that the unknown peak with an uncertain peak area appears in the sample, which indicates that the abnormal peak appearing in the solution is related to trichloromethane, but the peak area is uncertain.
In OOS2101001, a standard exceeding graph of impurity I and a normal sample detection graph (shown in figure 3) are compared, an abnormal peak of nifedipine impurity I comprises an unknown impurity peak between impurity I and impurity II, and a clear pollution source is not found in investigation.
In combination with OOT1605002 and OOS2101001, the two impurities are unknown impurities and I respectively, but the two abnormal conditions are similar to the peak type, so the two abnormal conditions are suspected to be the same reason, according to the investigation of OOT1605002, the root cause is unknown factors except chloroform, theoretically, the chloroform does not absorb ultraviolet rays, and does not follow the Lambert beer law, so the size of the peak area is not directly related to the amount of chloroform added, while the absorption peak of the chloroform generated in the sample is that the chloroform belongs to a fat-soluble solvent, is not completely miscible with methanol, generates bubbles or refraction effect to generate an absorption peak after being mixed with methanol, and after the sample is added, the refraction effect is enhanced to enlarge the absorption peak due to uncertain influences of grinding time, chloroform volatilization amount, auxiliary material filtration amount and the like, and the size is irregular and can be obtained due to not following the Lambert beer law, checking related substance detection methods, grinding after adding trichloromethane, transferring into a 100ml volumetric flask with methanol, adding methanol for grinding at first when a laboratory technician actually operates, because trichloromethane is very volatile, because different personnel, different environments, different operation habits and the like cause different volatilization degrees, the influence on impurity peaks is different, and the chromatogram of 9 test sample solutions detected at different times is shown in figure 2.
In summary, the reason why the abnormal peak appears here is that the chloroform and methanol added during the preparation of the sample solution will generate bubbles or refraction effect after mixing with the sample to generate an absorption peak.
Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.
Claims (10)
1. A nifedipine related substance analysis method based on HPLC is characterized in that related substances comprise an impurity I and an impurity II, the impurity I is nitropyridine, and the impurity II is nitrosopyridine, and the method mainly comprises the following steps:
preparing a test solution: grinding a test sample by adding trichloromethane, transferring the ground sample into a volumetric flask by using methanol, diluting the ground sample to obtain a test sample solution, and storing the test sample solution in a dark place;
preparation of control stock solutions: taking an impurity I reference substance and an impurity II reference substance, respectively adding methanol to dissolve and quantitatively diluting to prepare a mixed solution;
preparation of control solutions: taking a test solution and a reference stock solution, and quantitatively diluting with a mobile phase respectively;
system applicability solution: dissolving nifedipine, an impurity I reference substance and an impurity II reference substance in methanol and diluting to obtain system applicability solutions, wherein each 1ml of the system applicability solution contains 1mg, 10 mu g and 10 mu g of nifedipine, the impurity I reference substance and the impurity II reference substance respectively;
detecting the test solution and the control solution by a high performance liquid chromatograph;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-water (60:40) is used as a mobile phase, the detection wavelength is 235nm, and the sample injection volume is 20 mu l;
the determination method comprises the following steps: respectively injecting the test solution and the control solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 2 times;
limitation: if a chromatographic peak with the retention time consistent with that of the impurity I peak and that of the impurity II peak exists in a chromatogram of a test solution, the peak areas of the chromatographic peaks are not more than 0.1% by calculation according to an external standard method, the peak areas of other single impurities are not more than the peak area of nifedipine in a control solution, and the total amount of the impurities is not more than 0.5%.
2. The HPLC-based nifedipine-related substance analysis method of claim 1, wherein the specific method for preparing the test solution is as follows: grinding the sample with chloroform at 20-30 deg.C for 3-5 min at 60-150 r/min, transferring into volumetric flask with methanol, and diluting to obtain sample solution.
3. The HPLC-based nifedipine-related substance assay method of claim 1, wherein the control solution is prepared by the steps of: taking the sample solution and the reference stock solution, and quantitatively diluting with a mobile phase respectively to prepare a mixed solution containing 2 mu g of nifedipine, 1 mu g of impurity I and 1 mu g of impurity II in each 1 ml.
4. The HPLC-based nifedipine-related substance analysis method according to claim 2, wherein the grinding time is 3min, the grinding speed is 150r/min, and the grinding temperature is 30 ℃.
5. The HPLC-based nifedipine-related substance analysis method according to claim 2, wherein the grinding time is 4min, the grinding speed is 100r/min, and the grinding temperature is 25 ℃.
6. The HPLC-based nifedipine-related substance analysis method according to claim 2, wherein the grinding time is 5min, the grinding speed is 60r/min, and the grinding temperature is 20 ℃.
7. A nifedipine related substance analysis method based on HPLC is characterized in that related substances comprise an impurity I and an impurity II, the impurity I is nitropyridine, and the impurity II is nitrosopyridine, and the method mainly comprises the following steps:
preparing a test solution: grinding a test sample by adding trichloromethane, transferring the ground sample into a volumetric flask by using methanol, diluting the ground sample to obtain a test sample solution, and storing the test sample solution in a dark place;
preparing an alternative test solution: grinding the sample with chloroform at 20-30 deg.C for 3-5 min at 60-150 r/min, transferring into volumetric flask with methanol, and diluting to obtain sample solution;
preparation of control stock solutions: taking an impurity I reference substance and an impurity II reference substance, respectively adding methanol to dissolve and quantitatively diluting to prepare a mixed solution;
preparation of control solutions: taking a test solution and a reference stock solution, and quantitatively diluting by using a mobile phase respectively to prepare a mixed solution containing 2 mu g of nifedipine, 1 mu g of impurity I and 1 mu g of impurity II in each 1 ml;
system applicability solution: dissolving nifedipine, an impurity I reference substance and an impurity II reference substance in methanol and diluting to obtain system applicability solutions, wherein each 1ml of the system applicability solution contains 1mg, 10 mu g and 10 mu g of nifedipine, the impurity I reference substance and the impurity II reference substance respectively;
detecting the test solution and the control solution by a high performance liquid chromatograph;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol-water (60:40) is used as a mobile phase, the detection wavelength is 235nm, and the sample injection volume is 20 mu l;
the determination method comprises the following steps: respectively injecting the test solution and the control solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 2 times;
limitation: if a chromatographic peak with the retention time consistent with that of the impurity I peak and that of the impurity II peak exists in a chromatogram of a test solution, the chromatographic peak is not more than 0.1% when the chromatographic peak is calculated by an external standard method according to the peak area, the peak area of other single impurities is not more than the peak area of nifedipine in a control solution, and the total amount of the impurities is not more than 0.5%; and when the chromatographic peak which is consistent with the retention time of the impurity I peak is calculated by the peak area according to an external standard method and exceeds 0.1 percent, injecting the alternative sample solution into a liquid chromatograph, recording the chromatogram until the retention time of the main component peak is 2 times, and carrying out limit calculation again.
8. The HPLC-based nifedipine-related substance analysis method according to claim 7, wherein the alternative sample solution is prepared by grinding at a speed of 150r/min for 3min and at a temperature of 30 ℃.
9. The HPLC-based nifedipine-related substance analysis method according to claim 7, wherein the alternative sample solution is prepared by grinding at a speed of 100r/min for 4min and at a temperature of 25 ℃.
10. The HPLC-based nifedipine-related substance analysis method according to claim 7, wherein the alternative sample solution is prepared by grinding at a speed of 60r/min for 5min and at a temperature of 20 ℃.
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