CN116008450B - Method for detecting genotoxic impurities in pregabalin - Google Patents
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Abstract
The invention provides a method for detecting genotoxic impurities in pregabalin. The method comprises the following steps: (1) solution preparation: respectively preparing a sample solution and a reference solution by taking 1% formic acid solution as a solvent; (2) separation analysis: respectively injecting the sample solution and the reference substance solution into a high performance liquid chromatography-triple quadrupole tandem mass spectrometer to finish detection of genotoxic impurities; the mass spectrometer adopts an atmospheric pressure chemical ionization source and a multi-reaction monitoring mode, the high performance liquid chromatography adopts a C18 chromatographic column as a stationary phase, water-methanol as a mobile phase, and a gradient elution mode is adopted; the genotoxic impurities include cyanoacetamide, 2, 4-dicyano-3-isobutyl-glutaramide. The method has the advantages of high sensitivity, accuracy, reliability, good linear relation, and good precision and durability.
Description
Technical Field
The invention relates to the technical field of chromatographic detection, in particular to a method for detecting genotoxic impurities in pregabalin.
Background
Pregabalin (PGB) is a neurotransmitter gamma-aminobutyric acid analog; has good analgesic effect and less adverse reaction, is widely used for treating neuropathic pain such as postherpetic neuralgia and the like, and is recommended as a first-line therapeutic drug for a plurality of international guidelines.
The pregabalin production process is as follows:
the production process of the R-amide comprises the following steps:
The production process of the 3-isobutyl glutaric acid comprises the following steps:
As can be seen from the above synthetic route, the starting material for R-amide synthesis is cyanoacetamide, which requires ethyl cyanoacetate for synthesis, and is prepared by cyanation of ethyl chloroacetate with sodium cyanide, and the sodium cyanide used may remain in 3-isobutylglutaric acid. Acylation of isovaleraldehyde with cyanoacetamide may produce some cyano-containing byproducts, such as 2, 4-dicyano-3-isobutyl-glutaramide. Cyanoacetamide and 2, 4-dicyano-3-isobutyl-glutaramide are genotoxic impurities with a warning structure. The existing quality standards of pregabalin and capsules at home and abroad do not measure and control the cyanoacetamide and the 2, 4-dicyano-3-isobutyl-glutaramide. The publication does not report a trace of cyanoacetamide with 2, 4-dicyano-3-isobutyl-glutaramide assay.
The structural formula of the cyanoacetamide is as follows:
The structural formula of the 2, 4-dicyano-3-isobutyl glutaramide is as follows:
Two impurities in pregabalin drugs are genotoxic impurities with warning structures, namely, the two impurities can induce gene mutation and chromosome damage even cause cancerogenesis at a lower concentration level, have great safety risks, and have definite control guidelines and strict requirements for drug regulatory authorities in various countries. The clinical maximum daily dose of pregabalin is 600mg, calculated as the Threshold of Toxicological Concern (TTC) (maximum daily dose based on the pharmaceutical compound) ensuring a content of less than 1.5 μg/day, i.e. the product contains cyanoacetamide and 2, 4-dicyano-3-isobutyl-glutaramide in amounts which must be less than the following TTC levels: [ 1.5. Mu.g/day ]/600 mg=2.5 ppm. Therefore, there is a need for an accurate, reliable, highly sensitive assay for monitoring and mass analysis of cyanoacetamide and 2, 4-dicyano-3-isobutyl-glutaramide in pregabalin and its capsules.
CN110174467a discloses a method for analyzing and separating 2, 4-dicyano-3-isobutylglutarimide by high performance liquid chromatography. However, the method for measuring 2, 4-dicyano-3-isobutyl glutaramide is 2.5mg/ml, which far exceeds the set content of 1.5 mug, and the detection sensitivity is low, so that the requirement of the measurement limit of the corresponding impurities in the pregabalin medicine can not be met.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a method for detecting genotoxic impurities in pregabalin, which provides an accurate, reliable and high-sensitivity method for measuring the contents of cyanoacetamide and 2, 4-dicyano-3-isobutyl-glutaramide in pregabalin and capsules thereof, and overcomes the problem of low detection sensitivity in the prior art.
In one aspect of the present invention, a method for detecting genotoxic impurities in pregabalin is provided, comprising the steps of:
(1) Preparing a solution: respectively preparing a sample solution and a reference solution by taking 1% formic acid solution as a solvent;
(2) Separation analysis: respectively injecting the sample solution and the reference substance solution into a high performance liquid chromatography-triple quadrupole tandem mass spectrometer to finish detection of genotoxic impurities;
the mass spectrometer adopts an atmospheric pressure chemical ionization source and a multi-reaction monitoring mode, the high performance liquid chromatography adopts a C18 chromatographic column as a stationary phase, water-methanol as a mobile phase, and a gradient elution mode is adopted; the genotoxic impurities include cyanoacetamide, 2, 4-dicyano-3-isobutyl-glutaramide.
Further, the concentration of the test solution was 60mg/ml, and the concentration of the control solution was 150ng/ml.
Further, the test sample comprises pregabalin bulk drug and/or pregabalin capsule.
Further, the gas curtain gas pressure of the mass spectrum is 45psi; the collision gas pressure is 9psi; sprayer pressure was 50psi; the corona needle current was 4.0mA and the ion source temperature was 550 ℃.
Further, the ion mode adopted by the mass spectrum is a positive ion scanning mode; wherein, quantitative ion pairs are respectively: cyanoacetamide is m/z 85.1- > m/z42.0, 2, 4-dicyano-3-isobutyl-glutaramide is m/z 237.1- > m/z219.9; the qualitative ion pairs are respectively: cyanoacetamide was found to be m/z 237.1.fwdarw.m/z 85.1, and 2, 4-dicyano-3-isobutyl-glutaramide was found to be m/z 85.1.fwdarw.m/z 43.8.
Further, the C18 column had a particle diameter of 1.9 μm, a length of 150mm and an inner diameter of 2.0mm.
Preferably, the C18 column is YMC-Triart.
Further, the mobile phase A used for the liquid chromatography is water; mobile phase B was methanol.
Further, the flow rate of the mobile phase used for the liquid chromatography was 0.5ml/min.
Further, the column temperature used for the liquid chromatography is 40 ℃; the sample volume of the sample solution was 5. Mu.l.
Further, the elution gradient used for liquid chromatography was as follows:
the technical principle of the invention is as follows: the prior art generally detects the content of the single substance 2, 4-dicyano-3-isobutyl-glutaramide, because cyanoacetamide and 2, 4-dicyano-3-isobutyl-glutaramide have very different chemical properties and the measurement sensitivity of cyanoacetamide is low, so that the establishment of a method for simultaneously measuring two substances is very challenging. The inventor successfully establishes a method capable of simultaneously measuring the content of cyanoacetamide and 2, 4-dicyano-3-isobutyl-glutaramide in pregabalin and capsules thereof through repeated screening and detection of detection conditions such as a solvent, a mobile phase, an ion source and the like, and performs methodological verification.
Compared with the prior art, the invention has the following beneficial effects: the detection method of the cyanoacetamide and the 2, 4-dicyano-3-isobutyl-glutaramide in pregabalin provided by the invention has the advantages of high sensitivity, accuracy, reliability, good linear relation (R=0.9970 or R=0.9995), and good precision and durability. The recovery rate of the sample of the method is between 90.99 and 115.23 percent, and the accuracy is high. The detection limit of cyanoacetamide is 0.03ppm, and the quantitative limit is 0.1ppm; the detection limit of the 2, 4-dicyano-3-isobutyl glutaramide is 0.002ppm, the quantitative limit is 0.005ppm, and the detection sensitivity is high, thus being capable of being used as the basis of quality monitoring. In the detection method provided by the invention, the solvent does not interfere with detection of impurity cyanoacetamide and 2, 4-dicyano-3-isobutyl-glutaramide, the sample does not interfere with peak of impurity determination, the method has good specificity, and the method is simple, convenient, rapid, accurate and good in repeatability.
Drawings
FIG. 1 is a chromatogram of the XIC of 2, 4-dicyano-3-isobutyl-glutaramide in example 1 of the present invention.
FIG. 2 is a cyanoacetamide XIC chromatogram of the hollow white solvent of example 2 of the present invention.
FIG. 3 is a chromatogram of 2, 4-dicyano-3-isobutylglutarimide XIC in example 2 of the present invention as a white solvent.
FIG. 4 shows the cyanoacetamide XIC chromatogram of the hollow white vehicle of example 2 of the present invention.
FIG. 5 is a chromatogram of 2, 4-dicyano-3-isobutylglutarimide XIC of hollow white auxiliary material in example 2 of the present invention.
FIG. 6 is a cyanoacetamide XIC chromatogram of the control of example 2 of the present invention.
Fig. 7 is a chromatogram of pregabalin XIC of the control of example 2 of the present invention.
FIG. 8 is a chromatogram of 2, 4-dicyano-3-isobutylglutarimide XIC of the control in example 2 of the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and examples.
Example 1 determination of detection method
1. Instrument for measuring and controlling the intensity of light
UPLC-API5500 series triple quadrupole liquid chromatography-mass spectrometry (AB SCIEX company, USA); YMC-Triart C chromatographic column (150X 2.0mm,1.9 μm); XPE205 type electronic balance (Metrele Co.).
2. Reagent
R-amide (taicang madder, 20190703/20191104/20191105, cyanoacetamide control (Chongqing Saiwei pharmaceutical Co., ltd., lot No. 20200601, purity 99.8%), 2, 4-dicyano-3-isobutylglutamide control (Chongqing Saiwei pharmaceutical Co., ltd., lot No. 20200601, purity 99.7%); pregabalin samples (9 batches provided by Chongqing Sawye pharmaceutical Co., ltd.) and pregabalin capsule samples (20 batches provided by Chongqing Sawye pharmaceutical Co., ltd.).
Methanol and formic acid are all chromatographic purity; the water is ultrapure water.
3. Selection of method parameters
3.1 Selection of solvent
The pre-experiment shows that the cyanoacetamide has lower response, the signal to noise ratio is about 25-30 as measured by 15ng/ml solution sample injection, the concentration of a test sample is preferably as large as possible to meet the requirement of detection sensitivity, the inventor finds that the concentration requirement of a planned sample can be met in 1% formic acid solution by screening solvents such as water, methanol, acetonitrile, formic acid and the like, and the cyanoacetamide and the 2, 4-dicyano-3-isobutyl-glutaramide can also be dissolved, so that the 1% formic acid solution is selected as the solvent.
3.2 Selection of mobile phases
The positive ion mode of mass spectrometry typically adds formic acid to enhance ionization of the compound, enhance the response signal, and preliminary experiments show that acid in the mobile phase does not affect the determination of cyanoacetamide, but that the presence of very small amounts of acid is found to result in poor peaks of 2, 4-dicyano-3-isobutyl-glutaramide (see figure 1), possibly associated with the polybasic structure of the compound, and therefore water-methanol is selected as the mobile phase system.
3.3 Selection of ion sources
The electrospray ion source (ESI) is most commonly used, but the sensitivity is low, and the inventor finds that the sensitivity is obviously improved by adopting the atmospheric pressure chemical ionization source (APCI) through repeated experiments, so that the method can meet the requirement of low-response cyanoacetamide determination.
The specific method comprises the following steps: taking 15ng/ml cyanoacetamide solution, and adopting an electrospray ion source (ESI) and an atmospheric pressure chemical ionization source (APCI) to sample and measure respectively, wherein the signal to noise ratio of the cyanoacetamide is about 25-30 when the electrospray ion source (ESI) is adopted and the atmospheric pressure chemical ionization source (APCI) is adopted.
3.4 Establishment of the assay method
Preparation of a control solution: taking proper amounts of cyanoacetamide and 2, 4-dicyano-3-isobutyl-glutaramide reference substances, dissolving the cyanoacetamide and the 2, 4-dicyano-3-isobutyl-glutaramide reference substances by using 1% formic acid solution and preparing 150ng/ml solution.
Preparation of test solution: taking a proper amount of pregabalin sample, dissolving the pregabalin sample by using 1% formic acid solution, preparing a solution of 60mg/ml, centrifuging a capsule sample, and filtering the capsule sample through a 0.22 mu m filter membrane.
Chromatographic conditions: using YMC-Triart C chromatography column (150X 2.0mm,1.9 μm); taking water as a mobile phase A; methanol is used as a mobile phase B, and linear gradient elution is carried out according to the table 1; the flow rate is 0.5ml per minute; column temperature is 40 ℃; the sample loading was 5. Mu.l.
TABLE 1 gradient elution
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 85 | 15 |
| 0.5 | 85 | 15 |
| 3.0 | 75 | 25 |
| 6.0 | 50 | 50 |
| 8.0 | 50 | 50 |
| 8.1 | 85 | 15 |
| 13.0 | 85 | 15 |
Mass spectrometry conditions: a triple quadrupole tandem mass spectrometer is used as a detector, an atmospheric pressure chemical ionization source (APCI) is adopted, a positive ion scanning mode is adopted, and a monitoring mode is multi-reaction monitoring (MRM). The mass spectrum acquisition time is 0.5-1.0, 4.5-6.0 minutes, and pregabalin which is out of peak at about 3 minutes does not enter the mass spectrometer. The monitored ion pairs and ion source parameters of the compounds are shown in Table 2 and Table 3.
Table 2 monitoring ion pairs with compounds
TABLE 3 ion Source parameters
EXAMPLE 2 methodology investigation
1. Specificity test
Taking 1% formic acid solution as blank solvent solution; taking about 0.1g of blank auxiliary material of the pregabalin capsule, adding 1% formic acid solution for dissolution and dilution to 100ml, taking a proper amount of centrifugation, filtering supernatant fluid by a 0.22 mu m filter membrane, and taking a subsequent filtrate as the blank auxiliary material solution; the pregabalin sample, cyanoacetamide and 2, 4-dicyano-3-isobutylglutarimide reference substance were dissolved in 1% formic acid solution in an appropriate amount, and each 1ml of the mixture solution was quantitatively prepared as a mixture reference substance solution containing about 150ng of each. Taking the blank solvent solution, blank auxiliary material solution and mixed reference substance solution, sequentially injecting the blank solvent solution, the blank auxiliary material solution and the mixed reference substance solution into a liquid chromatograph according to the measurement method in the embodiment 1, and recording a chromatogram, wherein the result is shown in fig. 2-8.
The results show that neither the blank solvent nor the blank auxiliary material interfere with the measurement. In the chromatogram of the mixed reference substance, the cyanoacetamide, the pregabalin and the 2, 4-dicyano-3-isobutyl glutaramide sequentially show peaks, and the method has good specificity. According to the characteristics of mass spectrometry, a sectional monitoring window is adopted, and a flow path is switched, so that the pregabalin with high concentration does not enter a mass spectrometer, and pollution is avoided.
2. Linear range
Taking about 10mg of each of cyanoacetamide and 2, 4-dicyano-3-isobutyl glutaramide reference substance, precisely weighing, adding 1% formic acid solution for dissolution and quantitative dilution to prepare serial solutions containing 15ng, 25ng, 50ng, 100ng, 150ng, 250ng and 300ng in each 1ml, taking 5 μl of each of the serial solutions, sequentially and respectively injecting the serial solutions into a liquid chromatograph according to the measurement method in the example 1, recording a chromatogram, and carrying out linear regression on the cyanoacetamide and the 2, 4-dicyano-3-isobutyl glutaramide according to the mass concentration (C) corresponding to the peak area (A), wherein the result shows that the cyanoacetamide and the 2, 4-dicyano-3-isobutyl glutaramide respectively have good linear relations in the concentration ranges of 16.766-335.33 ng/ml and 16.615-332.3 ng/ml. See tables 4-5.
TABLE 4 Cyanoacetamide Linear Range
TABLE 5 linear range of 2, 4-dicyano-3-isobutylglutarimide
3. Accuracy test
Expressed as recovery (%) of cyanoacetamide, 2, 4-dicyano-3-isobutylglutaric acid amide.
3.1 Crude drug recovery test
About 0.3g of pregabalin sample (batch PE (II) 20210210) is precisely weighed and placed in a 5ml measuring flask, 9 parts of the sample are added into each 3 parts of the sample, and each 3 parts of the sample is respectively added with low, medium and high reference substance solutions of about 500ng, 750ng and 1250ng of each of the cyanoacetamide and the 2, 4-dicyano-3-isobutyl glutaramide, 1% formic acid solution is added for dissolution and dilution to scale, shaking is carried out, each of the samples is precisely measured by 5 μl, the measurement method in example 1 is adopted, and the result shows that the recovery rates of the cyanoacetamide and the 2, 4-dicyano-3-isobutyl glutaramide are all 81.09% -118.74%, and the table 6 is shown.
TABLE 6 results of crude drug recovery test
3.2 Recovery test of capsules
Weighing about 0.3g of capsule blank auxiliary materials, placing the capsule blank auxiliary materials into a 5ml measuring flask, adding about 9 parts of low, medium and high reference substance solutions of 500ng, 750ng and 1250ng of each of the cyanoacetamide and the 2, 4-dicyano-3-isobutyl glutaramide into each 3 parts of the capsule blank auxiliary materials, adding 1% formic acid solution for dissolving and diluting to scale, shaking uniformly, precisely weighing 5 μl, and measuring according to a preparation method, wherein the result shows that the recovery rates of the cyanoacetamide and the 2, 4-dicyano-3-isobutyl glutaramide are between 91.95% and 105.11%, and the result is shown in table 7.
TABLE 7 results of crude drug recovery test
As can be seen from tables 6 to 7, the average recovery rates of cyanoacetamide in pregabalin drug substance and pregabalin capsule were 90.99% and 95.63%, respectively; the average recovery rate of the 2, 4-dicyano-3-isobutyl glutaramide is 115.23 percent and 102.99 percent respectively, and the RSD is less than 10 percent, which shows that the recovery rate of the method is good and the accuracy of the method is high.
4. Solution stability investigation
Taking the same reference substance solution, a crude drug test sample solution added with a proper amount of the reference substance and a capsule test sample solution added with a proper amount of the reference substance, sampling at 0, 2,4, 8 and 12 hours respectively, injecting into a liquid chromatograph according to the measurement method in the embodiment 1, recording a chromatogram, evaluating the stability of the reference substance solution and the test sample solution by using the change of peak areas of cyanoacetamide and 2, 4-dicyano-3-isobutyl glutaramide, wherein the relative standard deviation (RSD%) of the peak areas is less than 10%, and the stability is stable within 12 hours, as shown in the table 8.
TABLE 8 results of solution stability investigation
5. Repeatability test
The amounts of cyanoacetamide and 2, 4-dicyano-3-isobutylglutarimide were calculated by taking a pregabalin sample (PE (II) 20210210) and a pregabalin capsule sample (lot 01210408) and measuring 6 parts in parallel according to the measurement method in example 1, and the results are shown in Table 9.
Table 9 repeatability test results (n=6)
As is clear from Table 9, the repeated measurement of 6 parts of the test solutions prepared in the same batch revealed that neither cyanoacetamide nor 2, 4-dicyano-3-isobutylglutamide was detected, indicating that the method was excellent in reproducibility.
6. Precision test
5 Μl of the mixed control solution (about 150 ng/ml) of cyanoacetamide and 2, 4-dicyano-3-isobutylglutarimide was precisely measured, 6 needles were continuously introduced, and the chromatogram was recorded to examine the Relative Standard Deviation (RSD) of the retention time (RT, min) and peak area. In order to ensure the accuracy of the measurement result, the precision of the sample injection is taken as an index of the applicability of the system and is incorporated into a planned measurement method. The results are shown in Table 10.
Table 10 sample injection precision test results
As is clear from Table 10, the control solution was continuously examined 6 times, the retention time RSD of cyanoacetamide was 0.22%, and the peak area RSD was 1.65%; the retention time RSD of 2, 4-dicyano-3-isobutylglutarimide was 0.52% and the peak area RSD was 2.65%; all are smaller than 10%, which indicates that the method has good precision.
7. Detection limit and quantitative limit test
About 10mg each of cyanoacetamide and2, 4-dicyano-3-isobutylglutarimide control was precisely weighed, 1% formic acid solution was added for quantitative dilution to prepare a detection limit solution and a quantitative limit solution of about 10ng and 15ng, respectively, per 1ml, and 5. Mu.l each was precisely measured, and the detection limit (S/N=3) and the quantitative limit (S/N=10) were calculated by injecting the mixture into a liquid chromatograph according to the measurement method in example 1, recording the chromatogram, and the measurement method was performed. The results are shown in Table 11.
Table 11 detection limit and quantitative limit measurement results
As is clear from Table 11, the detection limit of cyanoacetamide was 0.03ppm and the quantitative limit was 0.1ppm; the detection limit of 2, 4-dicyano-3-isobutylglutaric acid amide was 0.002ppm, and the quantitative limit was 0.005ppm. Can meet the detection requirements of cyano acetamide and 2, 4-dicyano-3-isobutyl glutaramide in pregabalin. The method is high in sensitivity.
8. Durability test
The flow rate (. + -. 5%) was varied, control solutions (15 ng/ml) were taken and samples were continuously taken 3 times and chromatograms were recorded. The results showed that the signal to noise ratio of the resulting cyanoacetamide to the peak of 2, 4-dicyano-3-isobutylglutarimide was greater than 10 after varying the flow rate. See table 12.
Table 12 durability test results
As can be seen from Table 12, the fine tuning chromatographic conditions had no effect on the detection of cyanoacetamide and 2, 4-dicyano-3-isobutylglutarimide, demonstrating that the method has good durability and is suitable for reagent application and popularization.
9. Sample content determination
The test methods of example 1 were performed using pregabalin drug substance samples and capsule samples, and the results are shown in table 13.
TABLE 13 sample measurement results
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The embodiment shows that the method has high sensitivity, accuracy, reliability, good linear relation, good precision and durability, is suitable for detecting the cyanoacetamide and the 2, 4-dicyano-3-isobutyl-glutaramide in pregabalin, and can be used as the basis for quality monitoring.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (6)
1. A method for detecting genotoxic impurities in pregabalin is characterized by comprising the following steps of: the method comprises the following steps:
(1) Preparing a solution: respectively preparing a sample solution and a reference solution by taking 1% formic acid solution as a solvent;
(2) Separation analysis: respectively injecting the sample solution and the reference substance solution into a high performance liquid chromatography-triple quadrupole tandem mass spectrometer to finish detection of genotoxic impurities;
the mass spectrometer adopts an atmospheric pressure chemical ionization source and a multi-reaction monitoring mode, the high performance liquid chromatography adopts a C18 chromatographic column as a stationary phase, water-methanol as a mobile phase, and a gradient elution mode is adopted; the genotoxic impurities include cyanoacetamide, 2, 4-dicyano-3-isobutyl-glutaramide;
the concentration of the sample solution is 60mg/ml, and the concentration of the reference solution is 150ng/ml;
The test sample comprises pregabalin bulk drug and/or pregabalin capsule;
the gas curtain gas pressure of the mass spectrum is 45psi; the collision gas pressure is 9psi; sprayer pressure was 50psi; the current of the corona needle is 4.0mA, and the temperature of the ion source is 550 ℃;
the mobile phase A used for the liquid chromatography is water; mobile phase B is methanol;
The flow rate of the mobile phase used for the liquid chromatography was 0.5ml/min.
2. The method for detecting genotoxic impurities in pregabalin according to claim 1, characterized in that: the ion mode adopted by the mass spectrum is a positive ion scanning mode; wherein, quantitative ion pairs are respectively: cyanoacetamide is m/z 85.1- > m/z42.0, 2, 4-dicyano-3-isobutyl-glutaramide is m/z 237.1- > m/z219.9; the qualitative ion pairs are respectively: cyanoacetamide was found to be m/z 237.1.fwdarw.m/z 85.1, and 2, 4-dicyano-3-isobutyl-glutaramide was found to be m/z 85.1.fwdarw.m/z 43.8.
3. The method for detecting genotoxic impurities in pregabalin according to claim 1, characterized in that: the particle size of the C18 chromatographic column is 1.9 mu m, the length is 150mm, and the inner diameter is 2.0mm.
4. A method for detecting genotoxic impurities in pregabalin according to claim 1 or 3, characterized in that: the C18 chromatographic column is YMC-Triart.
5. The method for detecting genotoxic impurities in pregabalin according to claim 1, characterized in that: the column temperature used for the liquid chromatography is 40 ℃; the sample volume of the sample solution was 5. Mu.l.
6. The method for detecting genotoxic impurities in pregabalin according to claim 1, characterized in that: the elution gradient used for liquid chromatography was as follows:
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