阅读说明：本技术 一种注射液中有关物质长春胺酸与阿朴长春胺酸的检测方法 (Method for detecting related substances vinblastic acid and apovincamine acid in injection ) 是由 刘景萍 刘全国 陈克领 麦发任 吴育强 郑国菊 王家 李党 于 2020-11-30 设计创作，主要内容包括：本发明提供一种注射液中有关物质长春胺酸与阿朴长春胺酸的检测方法,采用高效液相色谱法对长春西汀注射液中杂质长春胺酸与杂质阿朴长春胺酸进行定量鉴别,色谱条件为：色谱柱的填充剂为氰基硅烷键合硅胶,流速为0.5-0.7mL·min-1,柱温为30～34℃,以体积比为30～65∶65～70的0.1mol/L～0.25mol/L三乙胺溶液和乙醇为流动相。本发明利用高效液相色谱法测定长春西汀注射液中杂质长春胺酸与杂质阿朴长春胺酸的含量,该方法具有分离效果好、灵敏、准确等优点,保证了本品的质量稳定均一性及疗效；本发明的方法简单、专属性强、重现性好,有效的保障了长春西汀注射液的质量和疗效,具有很强的实用性。(The invention provides a method for detecting related substances, namely vinblastine and apovincamine acid, in an injection, quantitative identification is carried out on impurity, namely vinblastine and apovincamine acid, in the vinpocetine injection by adopting a high performance liquid chromatography, and the chromatographic conditions are as follows: the filler of the chromatographic column is cyano silane bonded silica gel, the flow rate is 0.5-0.7 mL/min < -1 >, the column temperature is 30-34 ℃, and triethylamine solution and ethanol which are 0.1-0.25 mol/L and have the volume ratio of 30-65: 65-70 are used as mobile phases. The method utilizes the high performance liquid chromatography to measure the content of the impurity vincamine acid and the impurity apovincamine acid in the vinpocetine injection, has the advantages of good separation effect, sensitivity, accuracy and the like, and ensures the quality stability, uniformity and curative effect of the product; the method is simple, has strong specificity and good reproducibility, effectively ensures the quality and the curative effect of the vinpocetine injection and has strong practicability.)

1. A method for detecting related substances, namely vinblastine acid and apovincamine acid in injection is characterized in that high performance liquid chromatography is adopted to quantitatively identify impurity, namely vinblastine acid and impurity, apovincamine acid in the vinpocetine injection, and the chromatographic conditions are as follows: the filler of the chromatographic column is cyano silane bonded silica gel, the flow rate is 0.5-0.7 mL/min < -1 >, the column temperature is 30-34 ℃, and triethylamine solution and ethanol which are 0.1-0.25 mol/L and have the volume ratio of 30-65: 65-70 are used as mobile phases.

2. The method for controlling the quality of vinblastine and apovincamine as impurities in vinpocetine injection as claimed in claim 1, wherein the detection solution is prepared by the following steps:

(1) preparing a test solution:

taking a vinpocetine injection sample, and taking a mobile phase as a diluent to prepare a test solution, wherein each 1ml of the test solution contains 0.8-1.2mg of vinpocetine.

(2) Impurity control solution:

taking the reference substance of impurity vincamine acid and impurity apovincamine acid, and taking mobile phase as diluent to prepare reference substance solution of impurity, wherein each 1ml contains 1.6-2.4 μ g of vincamine acid and 1.6-2.4 μ g of apovincamine acid;

(3) system applicability test solution:

taking a vinpocetine reference substance, and taking the impurity reference substance solution in the step (2) as a diluent to prepare a system applicability test solution, wherein each 1ml of the system applicability test solution contains 0.8-1.2mg of vinpocetine.

3. The method as claimed in claim 1, wherein the chromatography column is ECOSIL CN chromatography column.

4. The method as claimed in claim 1, wherein the chromatographic column is 4.6mm x 250mm, 5 μm.

5. The method for detecting related substances vinblastine and apovincamine acid in injection as claimed in claim 1, wherein the sample volume is 15-20 μ L.

6. The method as claimed in claim 1, wherein the flow rate is 0.6 mL-min-1.

7. The method as claimed in claim 1, wherein the detection wavelength is 280 nm.

8. The method for controlling the quality of vinblastine and apovincamine as impurities in vinpocetine injection as claimed in claim 2, wherein the detection solution is prepared by the following steps:

(1) preparing a test solution:

taking a vinpocetine injection sample, and taking a mobile phase as a diluent to prepare a test solution, wherein 1.0mg of vinpocetine is contained in each 1ml of the test solution.

(2) Impurity control solution:

taking the reference substance of impurity vincamine acid and impurity apovincamine acid, and taking mobile phase as diluent to prepare impurity reference substance solution, wherein each 1ml contains 2.0 μ g of vincamine acid and 2.0 μ g of apovincamine acid respectively;

(3) system applicability test solution:

and (3) taking a vinpocetine reference substance, and preparing a system applicability test solution by taking the impurity reference substance solution obtained in the step (2) as a diluent, wherein each 1ml of the vinpocetine reference substance contains 1.0mg of vinpocetine.

Technical Field

The invention relates to the field of medicine quality detection, in particular to a method for detecting related substances, namely vinblastic acid and apovincamine acid in injection.

Background

Vinpocetine (vinpocetin, abbreviated as VIN) has the chemical name: ethyl (13aS,13bS) -13 a-ethyl-2, 3,5,6,13a,13 b-hexahydro-1H-indole [3,2,1-de ] pyrido [3,2,1-ij ] [1,5] naphthyridine-12-carboxylic acid.

The product is firstly researched and developed by Cedeon Richter, Hungarian medicine company in 1978 to be marketed, and is mainly used for treating cardiovascular and cerebrovascular diseases, ischemic hypertensive encephalopathy, cerebral arteriosclerosis, cerebral ischemia, intermittent cerebral blood flow insufficiency, cerebral vasospasm, cerebral thrombosis, and brain diseases caused by aging.

The chemical structure of the product is as follows:

referring to related varieties in Chinese pharmacopoeia 2015 edition, referring to the quality standard, adopting a high performance liquid chromatography, and adopting an isocratic elution mode, wherein the known impurities calculate the content of the known impurities by peak area according to an external standard method, the unknown impurities measure the content of the unknown impurities of the product according to a main component self-contrast method, and meanwhile, as researches on synthesis starting materials, synthesis intermediates and inquired related impurities are added, the methodology research on related substances is carried out again on the basis of the detection method of the related substances in the original declared standard, and the research on the related substances methodology finds that the vinpocetine synthesized intermediate, namely vinpocetine, and the apovincamine acid, which is the impurity, cannot be separated in the original detection method of the related substances, so that a detection method is urgently needed to solve the problems.

Disclosure of Invention

In view of the above, the invention provides a method for detecting related substances, namely vinpocetine and apovincamine acid, in the injection, and the method is simple, has good separation degree, strong specificity and good repeatability, effectively ensures the quality and the curative effect of the vinpocetine injection, and has strong practicability.

The technical scheme of the invention is realized as follows:

a method for detecting related substances, namely vinblastine acid and apovincamine acid in injection adopts high performance liquid chromatography to quantitatively identify impurity, namely vinblastine acid and impurity, apovincamine acid in the vinpocetine injection, and the chromatographic conditions are as follows: the filler of the chromatographic column is cyano silane bonded silica gel, the flow rate is 0.5-0.7 mL/min < -1 >, the column temperature is 30-34 ℃, and triethylamine solution and ethanol which are 0.1-0.25 mol/L and have the volume ratio of 30-65: 65-70 are used as mobile phases.

Further, the preparation method of the detection solution comprises the following steps:

(1) preparing a test solution:

taking a vinpocetine injection sample, and taking a mobile phase as a diluent to prepare a test solution, wherein each 1ml of the test solution contains 0.8-1.2mg of vinpocetine, and the preferential concentration is 1.0 mg;

(2) impurity control solution:

taking the reference substance of impurity vincamine acid and impurity apovincamine acid, and taking mobile phase as diluent to prepare reference substance solution of impurity, wherein each 1ml contains 1.6-2.4 μ g of vincamine acid and 1.6-2.4 μ g of apovincamine acid, preferably 2.0 μ g;

(3) system applicability test solution:

taking a vinpocetine reference substance, and taking the impurity reference substance solution in the step (2) as a diluent to prepare a system applicability test solution, wherein each 1ml of the system applicability test solution contains 0.8-1.2mg of vinpocetine, and the preferential concentration is 1.0 mg.

Further, the chromatographic column is an ECOSIL CN chromatographic column.

Further, the specification of the chromatographic column is 4.6mm × 250mm, 5 μm.

Further, the detection wavelength is 280 nm.

Furthermore, the sample injection amount is 15-20 mu L.

Further, the flow rate was 0.6 mL. min-1.

Compared with the prior art, the invention has the beneficial effects that:

(1) the method utilizes the high performance liquid chromatography, adopts the combination of the specific formula flow and the specific chromatographic condition to determine the contents of the impurity vincamine acid and the impurity apovincamine acid in the vinpocetine injection, has the advantages of good separation effect, sensitivity, accuracy and the like, and ensures the quality stability, the uniformity and the curative effect of the product;

(2) the quality control method of the invention is simple, has strong specificity and good reproducibility, effectively ensures the quality and the curative effect of the vinpocetine injection and has strong practicability.

Drawings

FIG. 1 is a high performance liquid chromatogram (scan) of the reference substance of vinblastine impurity and apovincamine acid impurity in the content determination of vinpocetine injection;

FIG. 2 is a high performance liquid chromatogram (scan) of the sample containing vinblastic acid impurity and apovincamine acid impurity in the content determination of vinpocetine injection;

FIG. 3 is a linear relationship between the peak area and the concentration of vincamine acid;

FIG. 4 is a linear relationship between apovincamine acid area and concentration.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

The vinpocetine injection adopted by the invention is produced by Hainan cucurbita doll pharmaceutical industry group limited company.

Example 1

(I) determination of impurity vincamine acid and impurity apovincamine acid

(1) Precisely measuring a proper amount of vinpocetine injection, and quantitatively diluting the vinpocetine injection by using a mobile phase to prepare a solution containing 1.0mg of vinpocetine in each 1ml serving as a test solution;

(2) taking appropriate amount of each of the impurity vincamine acid and the impurity apovincamine acid as reference substances, precisely weighing, adding mobile phase for dissolving, and quantitatively diluting to obtain mixed solution containing 2 μ g of each of the impurity vincamine acid and the impurity apovincamine acid per 1ml as reference substance solution;

(3) taking 10mg of a vinpocetine reference substance, putting the vinpocetine reference substance into a 10ml measuring flask, dissolving the vinpocetine reference substance by using an impurity reference substance solution, diluting the vinpocetine reference substance to a scale, and shaking up the vinpocetine reference substance to be used as a system applicability test solution;

(4) and (3) injecting 20 mu l of system applicability test solution into a liquid chromatograph, recording a chromatogram, and sequentially obtaining an impurity VIII peak, an impurity IX peak and a vinpocetine peak, wherein the separation degrees among the peaks meet the requirement.

(5) And (3) absorbing the reference solution and the test solution, respectively injecting the reference solution and the test solution into a liquid chromatograph for determination, wherein if a chromatographic peak with the retention time consistent with that of the peak VIII and the peak IX of the impurity exists in the chromatogram of the test solution, the peak area of the chromatographic peak is calculated according to an external standard method, and the vincamine acid and the apovincamine acid have no more than 0.2% of the labeled amount.

The chromatographic conditions are as follows: the filler of the chromatographic column is cyano silane bonded silica gel (ECOSIL CN, 4.6mm multiplied by 250mm, 5 μm), the flow rate is 0.6mL min < -1 >, the column temperature is 30 ℃, the detection wavelength is 280nm, the sample injection amount is 20 μ L, and 0.25mol/L triethylamine solution and ethanol with the volume ratio of 35: 65 are used as mobile phases.

The high performance liquid chromatogram of the impurity vincamine acid and the impurity apovincamine acid reference substance in the content determination of the vinpocetine injection is shown in figure 1;

the high performance liquid chromatogram of the sample containing the impurity vincamine acid and the impurity apovincamine acid in the content determination of the vinpocetine injection is shown in figure 2.

In the step (one), the HPLC is examined for the following specific analysis:

1. linear relation

The test results show that, as shown in FIG. 3-4, the peak area and the concentration of the apovincamine acid are in a good linear relationship within the concentration range of 0.554-8.861 μ g/ml, and the peak area and the concentration of the apovincamine acid are in a good linear relationship within the concentration range of 0.441-7.056 μ g/ml.

2. Specificity

The peak of the degradation product and the main peak in each destructive test can be effectively separated, no degradation impurity is generated in the blank of the auxiliary material, and the measurement of related substances is not interfered. Under each damage condition, the vinpocetine injection originally developed and marketed sample and the developed sample material reach conservation and balance. The similarity of the peak purity of the main peak in each destructive test is more than 0.999, which indicates that the main peak is a single substance peak, and the specificity of the detection method is good.

Example 2-this example differs from example 1 in that a 0.25mol/L triethylamine solution and ethanol in a volume ratio of 30: 70 are used as mobile phases. The results show that the peak shapes of vinpocetine, vincamine acid and apovincamine acid are good.

Example 3-this example differs from example 1 in that a 0.1mol/L triethylamine solution and ethanol in a volume ratio of 1: 1 are used as mobile phases. The results show that the peak shapes of vinpocetine, vincamine acid and apovincamine acid are good.

Example 4-this example differs from example 1 in that the column temperature is 34 ℃. The results show that the peak shapes of vinpocetine, vincamine acid and apovincamine acid are good.

Example 5-this example differs from example 1 in that the flow rate was 0.5mL min-1. The results show that the peak shapes of vinpocetine, vincamine acid and apovincamine acid are good.

Example 6-this example differs from example 1 in that the flow rate was 0.7mL min-1. The results show that the peak shapes of vinpocetine, vincamine acid and apovincamine acid are good.

Comparative example 1-this comparative example differs from example 1 in that the column was replaced with an exform C18 column. The results show that the vincamine acid peak and the apovincamine acid peak interfere with each other.

Comparative example 2-this comparative example differs from example 1 in that the flow rate was 1mL min-1, and the results showed that the vincamine acid peak and apovincamine acid peak interfere with each other.

Comparative example 3-this comparative example differs from example 1 in that the column temperature was 28 ℃, and the results show that the vincamine acid peak and apovincamine acid peak interfere with each other.

Comparative example 4-this comparative example differs from example 1 in that a 0.2mol/L ammonium acetate solution and acetonitrile in a volume ratio of 40:60 are used as mobile phases. The results show that the vincamine acid peak and the apovincamine acid peak interfere with each other.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.