CN109765316B - Method for detecting levetiracetam from medicine - Google Patents
Method for detecting levetiracetam from medicine Download PDFInfo
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Abstract
The invention discloses a method for detecting levetiracetam (R-isomer) from levetiracetam injection, which adopts a high performance liquid chromatography method to carry out qualitative or/and quantitative detection on the R-isomer, wherein the detection conditions of the liquid chromatography comprise that: a chromatographic column: cellulose-tris (3, 5-dichlorophenyl carbamate) silica gel column; mobile phase: n-hexane, anhydrous ethanol; wherein the ratio of n-hexane (vol.%) to absolute ethyl alcohol (vol.%) is 90-70: 10-30; the mobile phase adopts isocratic elution. The method can effectively detect the levetiracetam in the levetiracetam injection and has specificity and stability indication capability. The levetiracetam injection can be directly diluted and then detected, and a sample does not need to be pretreated, so that convenience and rapidness are realized; the method has the advantages of detection limit of 0.003 percent and quantification limit of 0.005 percent, and is accurate and sensitive.
Description
Technical Field
The invention relates to the field of impurity detection in medicines, in particular to a method for detecting levetiracetam in medicines.
Background
Levetiracetam (Levetiracetam), the alias Levetiracetam, rivastigmine, and Levetiracetam, is a chemical in the form of white crystalline powder. The chemical name is (S) -alpha-ethyl-2-oxo-1-pyrrolidine acetamide with the molecular formula of C8H14N2O2And the molecular weight is 170.20900. Levetiracetam is an antiepileptic drug and is mainly used for the additive treatment of partial seizures of epilepsy patients of adults and children over 4 years old clinically.
As is known, the quality of the medicine is determined by the content of process impurities in the medicine, levetiracetam is a chiral compound and has a chiral center, the antiepileptic effect of R-isomer of levetiracetam is not obvious, and therefore, the medicinal curative effect of levetiracetam related medicines is greatly influenced by the content of the levetiracetam, and the content of the levetiracetam in the levetiracetam related medicines needs to be strictly controlled.
In USP40 raw material quality standard, the R-isomer of levetiracetam is controlled, and the limit is 0.8%; the quality standard of the injection is not controlled; the control limit of R-isomer in the import registration standard (JX20150256) of levetiracetam injection is 0.80%; it can be seen that the quality control standards of both standards are high, and the quality of the drug cannot be well controlled.
In order to perform better quality control on the medicines, the quality of the final product needs to be detected in addition to the detection and analysis of the raw medicines, so that the quality of the medicines put into use can be really controlled.
In USP40 raw material quality standard and levetiracetam injection import registration standard, used chromatographic columns are all silica gel columns coated with amylose tri-3, 5 dimethyl phenyl carbamate porous spherical columns, the chromatographic columns cannot contact water, if the chromatographic columns are used for levetiracetam injection detection, the column efficiency of the chromatographic columns can be rapidly reduced, only the samples of the levetiracetam injection can be subjected to fussy pretreatment, and after water in the samples is removed, the quality detection is carried out by using a method for detecting various substances in levetiracetam raw material medicines, so that the method is very troublesome.
Therefore, even if the technology is improved to reduce the quality control standard limit of the levetiracetam raw material medicine and improve the quality control technology of the levetiracetam raw material medicine, the problem of how to conveniently and accurately perform good quality control on the levetiracetam injection cannot be fundamentally solved.
Disclosure of Invention
The invention provides a method for detecting levetiracetam, which can accurately, conveniently and sensitively determine the content of levetiracetam in levetiracetam bulk drugs or injection, and the separation degree meets the regulation.
Specifically, the invention provides a method for detecting levetiracetam, which is characterized in that high performance liquid chromatography is adopted for qualitative or/and quantitative detection, and the detection conditions of the liquid chromatography comprise:
a chromatographic column: cellulose-tris (3, 5-dichlorophenyl carbamate) silica gel column;
mobile phase: n-hexane, anhydrous ethanol;
wherein, the normal hexane (vol.%) is 82-78: 18-22; the mobile phase adopts isocratic elution.
The isocratic elution refers to an elution mode in which the composition ratio and the flow rate of a mobile phase are constant in an analysis period of a sample component in the liquid chromatography operation.
In one embodiment of the invention, n-hexane (vol.%): absolute ethanol (vol.%) in the mobile phase is 80: 20.
In a particular embodiment of the invention, the liquid chromatography detection conditions further comprise one or more of the following i to iv:
i specification of chromatographic column: 4.6 x 100-250 mm, 5 μm;
ii flow rate: 0.5-1.5 ml/min;
iii column temperature: 25-35 ℃;
iv detection wavelength: 210-220 nm.
Further, the liquid chromatography detection conditions further comprise one or more of the following i to iv:
i specification of chromatographic column: 4.6X 250mm, 5 μm;
ii flow rate: 1.0 ml/min;
iii column temperature: 30 ℃;
iv detection wavelength: 215 nm.
The column used in the embodiment of the present invention is CHIRALPAKIC,250 mm. times.4.6 mm, 5 μm, but a column equivalent to the above column may also be used in the detection method of the present invention.
Further, the detection method comprises the following steps:
(1) preparing a test solution;
(2) preparing a reference solution;
(3) injecting a reference substance solution for detection;
(4) and (5) injecting a sample solution for detection.
The detection method can analyze and calculate the detection result by using methods such as an area normalization method, a self-comparison method, an internal standard method, an external standard method and the like.
Further, the step of preparing the test solution or/and the control solution is: dissolving a sample by using a solvent, and diluting the sample to a specified concentration by using a diluent; or directly dissolving and diluting the sample to a specified concentration by using a diluent.
Further, the solvent is one or a mixture of water, absolute ethyl alcohol, a sodium chloride solution and an acetic acid-sodium acetate buffer solution; the diluent is selected from absolute ethyl alcohol.
Furthermore, the concentration of the test solution is 5-15 mg/ml, preferably 10 mg/ml.
Furthermore, the concentration of the control solution is 10-50 mug/ml, preferably 20 mug/ml.
Furthermore, the sample amount is 10 to 50 μ l, preferably 20 μ l.
The method can independently control the quality of the levetiracetam injection, and simultaneously, as the chromatographic column can be directly used for detecting the water-containing test sample, the chromatographic column is shared with the quality detection of the bulk drug in the drug quality control, so that the quality control of the bulk drug and the injection can be quickly and conveniently carried out.
The invention also provides levetiracetam injection, which comprises levetiracetam and impurities, wherein the impurities comprise R-enantiomer of the levetiracetam, and the content of the R-enantiomer is not more than 0.2%.
The invention has the beneficial effects that:
(1) the invention provides a method for directly detecting R-enantiomer impurities of levetiracetam injection, which fills the blank of a levetiracetam injection quality control method, provides a further control method for levetiracetam medicine quality, and is beneficial to improving medicine quality.
(2) The detection method can effectively detect the R-isomer in the levetiracetam related medicine, has specificity and stability indication capability, can be effectively separated from the levetiracetam main peak, has good durability, and can well meet the requirements of impurity monitoring in the process and impurity control in levetiracetam related finished products.
(3) The method can directly dilute the levetiracetam injection for detection, does not need to pretreat water contained in the levetiracetam injection, has good peak shape of the obtained detection spectrogram and no tailing, is more convenient and rapid than the prior art, and is the first method for controlling the quality of the levetiracetam injection.
(4) The analysis method has high sensitivity, can strictly control the R-isomer of levetiracetam, has the detection limit of 0.003 percent and the quantification limit of 0.005 percent, is accurate and sensitive, can control the isomer limit to be less than 0.2 percent, and can better control the quality of the medicine.
Drawings
FIG. 1 is a blank solvent, system applicability, and sample overlay under chromatographic conditions in accordance with the present invention;
FIG. 2 is a detection spectrum of a blank solvent under chromatographic conditions according to the present invention;
FIG. 3 is a detection spectrum of blank auxiliary materials under chromatographic conditions according to the present invention;
FIG. 4 is a detection spectrum of levetiracetam racemate under chromatographic conditions according to the invention;
FIG. 5 is a detection spectrum of levetiracetam control under chromatographic conditions in accordance with the present invention;
FIG. 6 is a detection spectrum under chromatographic conditions of the invention;
FIG. 7 is a chromatogram of levetiracetam racemate using an AD-H chromatographic column
FIG. 8 is a chromatogram of a sample using an AD-H column (primary measurement injection).
FIG. 9 shows a chromatogram obtained by superposing a racemate, a control and a sample on an AD-H column (a re-measurement of an injection).
Detailed Description
Example 1
Blank solution: anhydrous ethanol.
Blank auxiliary material stock solution: preparing a blank solution without levetiracetam raw material medicines according to the prescription amount; about 4.5g of sodium chloride, about 0.82g of sodium acetate and about 0.6ml to 500ml of 5% acetic acid are weighed in the water for injection.
Blank adjuvant solution: precisely measuring the blank auxiliary material stock solution in a measuring flask of 1.0ml to 10ml, and diluting the blank auxiliary material stock solution to a scale by using absolute ethyl alcohol to obtain the finished product.
System applicability solution: weighing 11.60mg of levetiracetam racemate reference substance, placing the reference substance into a 100ml measuring flask, dissolving the reference substance by using absolute ethyl alcohol, and diluting the reference substance to a scale to prepare a solution containing about 0.1mg in each 1ml, thus obtaining the levetiracetam.
Levetiracetam control solution: accurately weighing 11.03mg of levetiracetam reference substance, placing the levetiracetam reference substance in a 200ml measuring flask, dissolving the levetiracetam reference substance by using absolute ethyl alcohol, diluting the levetiracetam reference substance to a scale mark, and preparing a solution containing 50 mu g/ml in each 1ml to obtain the levetiracetam.
Test solution: taking 1.0ml of levetiracetam injection, placing the levetiracetam injection into a 10ml measuring flask, diluting the levetiracetam injection with absolute ethyl alcohol to a constant volume to scale, and preparing a solution containing 10mg/ml in each 1ml to obtain the levetiracetam injection.
The detection method for the R-isomer in USP40 and the imported registration standards was compared. The quality standards of USP40 injection are compared because the isomers are not controlled in the quality standard (see Table 1).
TABLE 1 comparison of isomer quality standards
The USP40 and imported registration standard chromatographic column represents CHIRALPAK AD-H (250mm multiplied by 4.6mm, 5 mu m) model, the chromatographic column can not contact water, and if the method is used for detecting levetiracetam injection, the sample pretreatment process is too complicated, so the chromatographic column is screened.
Since the column CHIRALPAK IC (250 mm. times.4.6 mm, 5 μm) was used for both the normal phase and the reverse phase, the column was selected for testing under the same chromatographic conditions. The experimental results show that: as shown in FIG. 3, the R-isomer appeared after the main peak, and the degree of separation was satisfactory. The condition is determined as a detection method of levetiracetam injection.
Example 2 analytical method validation
The invention preliminarily establishes chromatographic conditions of the R-isomer analysis method, and the detection result shows that the method can effectively separate the R-isomer from the main drug.
The specific verification results are shown in table 2:
TABLE 2 verification summary of R-isomer analysis method of levetiracetam injection
The results show that the system applicability and specificity, the detection limit and the quantification limit, the linearity and range, the precision, the accuracy and the durability of the method of the detection method all meet the requirements, and the detection method is proved to be suitable for the detection of the R-isomer of levetiracetam.
The technical effects of the present invention are further illustrated by comparison with the following control tests:
comparative example 1
The levetiracetam injection is detected by adopting the chromatographic conditions of USP40 and using CHIRALPAK AD-H (250mm multiplied by 4.6mm, 5 mu m) as a chromatographic column, the preparation method of a test sample is the same as that of example 1, the detection result is shown in figures 7 and 8, the peak emergence time of levetiracetam is later than that of R-isomer of levetiracetam, the peak shape is better when the injection is measured for the first time, and the levetiracetam and the R-isomer of levetiracetam can be effectively separated. However, the chromatographic column cannot contact water, and the levetiracetam injection is subsequently detected by adopting the chromatographic column, the detection result is shown in fig. 9, and the result shows that when a sample to be detected contains water, the column efficiency of the chromatographic column is rapidly reduced, the trailing of each peak is serious, the peak type is poor, the R-isomer content cannot be accurately judged, the chromatographic column is expensive, the loss is accelerated, and the test cost is increased.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (8)
1. The detection method of the R-enantiomer in the levetiracetam injection is characterized in that high performance liquid chromatography is adopted for qualitative or/and quantitative detection, and the detection conditions of the liquid chromatography comprise:
a chromatographic column: cellulose-tris (3, 5-dichlorophenyl carbamate) silica gel column; mobile phase: n-hexane, anhydrous ethanol;
wherein the volume ratio of n-hexane to absolute ethyl alcohol is 90-70: 10-30; isocratic elution is adopted for the mobile phase;
the detection method comprises the following steps:
(1) preparing a test solution;
(2) preparing a reference solution;
(3) injecting a reference substance solution for detection;
(4) sample introduction and detection are carried out on the test solution;
the steps for preparing the test solution or/and the control solution are as follows: dissolving a sample by using a solvent, and diluting the sample to a specified concentration by using a diluent; or directly dissolving and diluting the sample to a specified concentration by using a diluent; the solvent is one or more selected from water, absolute ethyl alcohol, sodium chloride solution and acetic acid-sodium acetate buffer solution; the diluent is selected from absolute ethyl alcohol.
2. The detection method according to claim 1, wherein the ratio of n-hexane to absolute ethanol in the mobile phase is 80:20 by vol.%.
3. The detection method according to claim 1, wherein the liquid chromatography detection conditions further comprise one or more of the following i to iv:
i specification of chromatographic column: 4.6 x 100-250 mm, 5 μm;
ii flow rate: 0.5-1.5 ml/min;
iii column temperature: 25-35 ℃;
iv detection wavelength: 210-220 nm.
4. The detection method according to claim 3, wherein the liquid chromatography detection conditions further comprise one or more of the following i to iv:
i specification of chromatographic column: 4.6X 250mm, 5 μm;
ii flow rate: 1.0 ml/min;
iii column temperature: 30 ℃;
iv detection wavelength: 215 nm.
5. The detection method according to claim 1, wherein the concentration of the sample solution is 5 to 15 mg/ml; the concentration of the reference substance solution is 10-50 mug/ml.
6. The detection method according to claim 5, wherein the concentration of the test solution is 10 mg/ml; the concentration of the control solution is 20 mug/ml.
7. The detection method according to claim 1, wherein the amount of the sample is 10 to 50. mu.l.
8. The detection method according to claim 7, wherein the amount of the sample is 20. mu.l.
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| CN101532996A (en) * | 2008-03-10 | 2009-09-16 | 北京德众万全医药科技有限公司 | Method for analyzing and separating levetiracetam by using HPLC method |
| CN105987961A (en) * | 2015-02-04 | 2016-10-05 | 四川大学华西医院 | Detection method of levetiracetam in breast milk |
| CN107167401A (en) * | 2017-05-18 | 2017-09-15 | 湖南洞庭药业股份有限公司 | Levetiracetam sustained-release tablets pharmaceutical composition and its Quality Control and preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101532996A (en) * | 2008-03-10 | 2009-09-16 | 北京德众万全医药科技有限公司 | Method for analyzing and separating levetiracetam by using HPLC method |
| CN105987961A (en) * | 2015-02-04 | 2016-10-05 | 四川大学华西医院 | Detection method of levetiracetam in breast milk |
| CN107167401A (en) * | 2017-05-18 | 2017-09-15 | 湖南洞庭药业股份有限公司 | Levetiracetam sustained-release tablets pharmaceutical composition and its Quality Control and preparation method |
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| HPLC 法测定左乙拉西坦片的右旋异构体;付建等;《山东化工》;20141231;第2-3部分 * |
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