CN110530993B - Improved detection method of deacetylated hairy glycoside related substance - Google Patents
Improved detection method of deacetylated hairy glycoside related substance Download PDFInfo
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Abstract
The invention discloses an improved analysis method for detecting related substances of deacetyl hairy flower glycoside, which comprises the following steps of adopting a reversed phase C18 column and taking water as a mobile phase A; the volume ratio is 20: 16-24: 14, taking acetonitrile-methanol as a mobile phase B; the temperature of the chromatographic column is 25-35 ℃, the detection wavelength is 200-270 nm, the flow rate is 0.8-1.5 ml/min, and gradient elution is carried out.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a method for analyzing and detecting a medicine, and particularly relates to a method for detecting deacetyl hairy flower glycoside and related substances thereof.
Background
In the research and development of new drugs or imitation drugs, impurities are key quality attributes affecting products. The structure, physicochemical property and toxicity of impurities are complex, so the examination and research of related substances are one of the difficulties in drug development. The adverse reaction generated in the clinical use process of the medicine is not only related to the pharmacological activity of the main component, but also is closely indistinguishable from the influence of impurities. In order to ensure safe clinical medication, impurities in the medicine must be strictly controlled.
Deacetyl hairy flower glycoside, EnglishThe name of the product is Deslanoside, the chemical name is 3- [ [ O-beta-D-glucopyranosyl- (1 → 4) -O-2, 6-dideoxy-beta-D-ribo-hexopyranosyl]Oxo radical]-12, 14-dihydroxy-cardol-20 (22) -enolide with molecular formula of C47H74O19Molecular weight 943.09, CAS number 17598-65-1, structural formula as follows:
an injection of desacetylhairy flower glycoside, which is sold as cedilan, is an antiarrhythmic drug and is mainly used for heart failure. Because of its fast action, it is suitable for patients with acute cardiac insufficiency or acute exacerbation of chronic cardiac insufficiency, and can be used for controlling the ventricular rate of patients with atrial fibrillation and atrial flutter accompanied by fast ventricular rate, and sometimes for stopping supraventricular tachycardia and its slow action.
The related substances in the desacetylhairoside injection can be derived from two aspects, process impurities and degradation impurities, wherein the structures of part of the process impurities and the degradation impurities are determined as shown in table 0:
table 0: summary of deacetyl hairy glycoside hybrid mass spectrometry
According to the literature search, in the methods for detecting the deacetylated hairy flower glycoside and related substances, the thin-layer chromatography is adopted in foreign pharmacopoeias such as European pharmacopoeia and Japanese pharmacopoeia, and the content of the related substances is difficult to accurately detect. The method of the Chinese pharmacopoeia 2015 adopts a high performance liquid chromatography method, and uses octadecylsilane chemically bonded silica as a filling agent; taking water as a mobile phase A; acetonitrile-methanol (22: 14) is used as a mobile phase B; the detection wavelength was 220nm and the gradient elution was performed as in Table 1.
TABLE 1 examination method of related substance of deacetyl hairy flower glycoside
The high performance liquid chromatography of the Chinese pharmacopoeia 2015 for detecting related substances of the desacetyleriocitrin is more accurate than the thin-layer chromatography, but still has some defects, such as incapability of detecting all impurities, poor separation degree, limited separation capacity and detection capacity of the impurities, and further improvement on the quality control of products. Therefore, a more applicable detection method for the deacetylated hairy flower glycoside related substances is urgently needed to be found.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides an improved method for detecting the deacetyl hairy flower glycoside and related substances, and the method for separating and determining the deacetyl hairy flower glycoside related substances in the patent application solves the technical problems that the method for determining the deacetyl hairy flower glycoside related substances in the prior art is low in detection capability and inaccurate.
The invention provides an improved method for separating and measuring related substances of deacetyl hairy flower glycoside, which comprises the following steps: adopting a reversed-phase high performance liquid chromatograph; adopting a reversed phase C18 column, and taking water as a mobile phase A; and (2) mixing the components in a volume ratio of 20: 16-24: performing gradient elution by using acetonitrile-methanol of 14 as a mobile phase B; the temperature of the chromatographic column is 25-35 ℃, the detection wavelength is 200-270 nm, and the flow rate is 0.8-1.5 ml/min; (ii) a
Preferably, the gradient elution is preferably as follows:
0-25 min, and 67% of mobile phase A; 26-26 min, reducing the mobile phase A from 67% to 60%; 26-38 min, wherein the mobile phase A is 60%; reducing the content of the mobile phase A from 60% to 45% in 38-39 min; 39-50 min, wherein the content of the mobile phase A is 45%, and 50-51 min, the content of the mobile phase A is increased from 45% to 67%; then the equilibration continued for 5min at 67% mobile phase a.
Preferably, the column is Agilent ZORBAX SB C18, more preferably, the inner diameter of the column is 4.6mm x 250mm and the packing particle size is 3.5 μm.
Preferably, the column temperature is 30 ℃.
Preferably, the detection wavelength is 220 nm.
Preferably, the flow rate is 1.0 ml/min.
Preferably, the mobile phase B is a mixture of 20: 16 solution of acetonitrile and methanol.
The invention has the following beneficial effects: the novel detection method for related substances of the desacetylhairoside has the advantages of high detection rate of related substances, high precision, good repeatability and recovery rate, control over 5 key known impurities and 5 key unknown impurities, guarantee of the quality of the desacetylhairoside and final products of injection thereof, and implementation of the concept that the quality is derived from design in actual production. The method can detect more impurities, particularly can detect the impurities C and E, can ensure the separation degree among peaks, and sets the limit of key impurities according to the detection results of each batch during detection, thereby greatly improving the economic applicability in the actual production process.
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FIG. 1 is a map of the applicability of the system of example 1 of the present invention;
FIG. 2 is a graph of a test solution of example 1 of the present invention;
FIG. 3 is a map of comparative example 1 of the present invention.
Detailed Description
The following embodiments of the present invention will be described in further detail with reference to the accompanying drawings, and it should be understood that the embodiments are described for better understanding of the technical scheme of the present application, but not for limitation of the technical scheme of the present application, and any combination of the above impurities and the deacetylated hairy flower glycoside isolated and determined by the method of the present invention falls within the scope of the present invention.
Example 1 System applicability
The instrument comprises the following steps: waters e2695/2489 high performance liquid chromatograph
A chromatographic column: agilent ZORBAX SB C18(4.6 mm. times.250 mm, 3.5 μm)
Mobile phase A: water (W)
Mobile phase B: acetonitrile-methanol (20: 16, v/v)
The flow rate is 1.0 ml/min;
the temperature of the chromatographic column is 30 ℃;
the detection wavelength is 220 nm;
the sample amount is 20 mul;
gradient elution was performed according to the gradient shown in table 2:
TABLE 2 gradient elution
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 67 | 33 |
| 25 | 67 | 33 |
| 26 | 60 | 40 |
| 38 | 60 | 40 |
| 39 | 45 | 55 |
| 50 | 45 | 55 |
| 51 | 67 | 33 |
| 56 | 67 | 33 |
Preparing a system applicability test solution: precisely weighing appropriate amount of each of the deacetyl hairy flower glycoside, the impurity F reference substance, the impurity G reference substance, the impurity H reference substance, the impurity C reference substance and the impurity E reference substance, dissolving the solution in a solvent, and diluting to prepare a solution containing about 0.2mg of the deacetyl hairy flower glycoside, about 7 mu G of the impurity F, about 4 mu G of the impurity G, H and about 1 mu G of the impurity C, E per 1ml of the solution to be used as a system applicability test solution.
Blank solution (diluent): acetonitrile: methanol: water 232: 148: 620 (volume ratio).
Preparing a test solution: collecting the injection containing desacetylhairoside.
Preparation of a control solution: precisely measuring 1ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to the scale, and shaking up to obtain a control solution (1%).
And (3) determination: respectively taking blank solvent, system applicability solution, control solution and sample solution 20 μ l, respectively, injecting into high performance liquid chromatograph, and recording chromatogram. The results are as follows: see table 3, system suitability map see fig. 1; the profile of the control solution is shown in FIG. 2
TABLE 3 Deacetyllanatoside and related substance separation
As a result: the blank solvent has no interference to the determination, and the peak emergence order is impurity F peak, impurity G peak, deacetyl hairy flower glycoside peak, impurity H peak, impurity C peak and impurity E peak. The minimum separation degree between each impurity peak and the main component of the deacetyl hairy flower glycoside and between each impurity peak is 2.01 (the separation degree is more than or equal to 1.5); it follows that the results meet the system suitability requirements.
Example 2 detection and quantitation limits
Experimental conditions, liquid chromatography methods and solution preparation As shown in example 1, solutions can also be prepared according to methods known to those skilled in the art.
For known impurities: diluting the impurity solution with known concentration and the deacetyl hairy glycoside solution to lower concentration, comparing the measured signal with a baseline signal (baseline noise), and taking the corresponding concentration of the known impurity with the signal-to-noise ratio s/n responding to an instrument being more than or equal to 10 as the quantitative limit of the method; the corresponding concentration of the lowest response in the known impurities at the signal-to-noise ratio s/n of more than or equal to 3 on the instrument is the detection limit of the method.
For unknown impurities: the main component of the deacetyl hairy flower glycoside is used for replacing the quantitative limit concentration and the detection limit concentration for examining a single unknown impurity.
The test results are shown in Table 4.
TABLE 4 determination of detection limit and quantitation limit for impurity F
The detection limit of the impurity F is 0.0094 mu g/ml, and the quantification limit is 0.0312 mu g/ml; the detection limit of the impurity G is 0.0225 mug/ml, and the quantification limit is 0.0749 mug/ml; the detection limit of the deacetyl hairy flower glycoside is 0.0587 mu g/ml, and the quantification limit is 0.1956 mu g/ml; the detection limit of the impurity H is 0.0587 mu g/ml, and the quantification limit is 0.1955 mu g/ml; the detection limit of the impurity C is 0.0573 mug/ml, and the quantification limit is 0.1910 mug/ml; the detection limit of the impurity E is 0.0587 mu g/ml, and the quantification limit is 0.1956 mu g/ml.
Example 3 linearity
Experimental conditions, liquid chromatography methods and solution preparation As in example 1, solutions can also be prepared according to methods well known to those skilled in the art.
For known impurities, 6 concentration points were taken in the range from the LOQ (limit of quantitation of LOQ) concentration to the index concentration of not less than 150% and studied. The linear relationship is plotted as a function of the measured response signal (peak area) versus analyte concentration, and a linear regression is performed using a least squares method, with at least the correlation coefficient R being reported2To verify a good linear relationship, it is required that the coefficient should be between 0.990 and 1.000.
For unknown impurities, the linearity and range of the unknown impurities are examined by using a deacetyl hairy flower glycoside sample instead.
The linear relationship and the results of the analysis of each substance are shown in tables 5 to 10
1. Impurity F
TABLE 5 Linear measurement of impurity F
The linear equation is Y-43634.2577X + 521.6178; the linear regression coefficient R is in the range of 0.0312 mug/ml to 12.4640 mug/ml21.0000, good linearity;
2. impurity G
TABLE 6 Linear measurement of impurity G
The linear equation is Y-33860.3698X-139.3229; linear regression coefficient R in the range of 0.0749-7.4940 mug/ml21.0000, good linearity;
3. deacetylhairy flower glycoside
TABLE 7 Linear assay of Deacetyllanatoside
The linear equation is Y-18434.3624X-101.6787; linear regression coefficient R in the range of 0.1956-7.8780 mug/ml2Is 1.0000, the linearity is good.
4. Impurity H
TABLE 8 Linear measurement of impurity H
The linear equation is Y-26880.9022X-124.0079; linear regression coefficient R in the range of 0.1955-7.8200 mug/ml2Is 1.0000, the linearity is good.
5. Impurity C
TABLE 9 Linear measurement of impurity C
The linear equation is Y-16753.1000X-18.1579; linear regression coefficient R in the range of 0.1910-1.9100 mug/ml20.9999, the linear relationship is good.
6. Impurity E
TABLE 10 Linear measurement of impurity E
The linear equation is Y-22687.4996X + 86.7337; linear regression coefficient R in the range of 0.1956-1.9560 mug/ml2Is 1.0000, the linearity is good.
Comparative example 1: chromatographic method for measuring related substances of deacetyl hairy flower glycoside injection in Chinese pharmacopoeia 2015 edition
Detection solution: the test solution of example 1 was used
The instrument comprises the following steps: waters e2695/2489 high performance liquid chromatograph
A chromatographic column: agilent ZORBAX SB C18(4.6 mm. times.250 mm, 3.5 μm)
Mobile phase A: water (W)
Mobile phase B: acetonitrile-methanol (22: 14, v/v)
The flow rate is 1.0 ml/min; the column temperature is 30 ℃; the detection wavelength is 220 nm; the sample amount is 20 mul;
gradient elution, the elution procedure is: as set forth in the table below,
as shown in a chromatogram recorded in figure 3, the peak position of the impurity C, E in the method is on the slope, the impurity cannot be detected, the unknown impurities in F, G and 6.282min are very close, and when the chromatographic conditions are slightly changed, the separation degree of the impurities is poor, so that the detection result is influenced.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such changes and modifications of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such changes and modifications.
Claims (4)
1. An improved detection method of related substances of deacetyl hairy flower glycoside is characterized by comprising the following steps of adopting a reversed-phase high performance liquid chromatograph; the temperature of a chromatographic column is 25-35 ℃, the detection wavelength is 200-270 nm, the flow rate is 0.8-1.5 ml/min, a reversed-phase C18 chromatographic column is adopted, and the reversed-phase C18 chromatographic column is Agilent ZORBAX SB C18, 4.6mm is multiplied by 250mm, and 3.5 mu m; taking water as a mobile phase A; the volume ratio is 20: performing gradient elution by using 16 acetonitrile-methanol as a mobile phase B; the gradient elution was as follows:
；
The related substances comprise five known impurities and any one or more of five unknown impurities, and the five known impurities have the following structures:
2. the method of claim 1, wherein the column temperature is 30 ℃.
3. The detection method according to claim 1, wherein the detection wavelength is 220 nm.
4. The assay of claim 1, wherein the flow rate is 1.0 ml/min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961326A (en) * | 2012-11-27 | 2013-03-13 | 上海朝晖药业有限公司 | Preparation method of deslanoside injection |
| CN104447933A (en) * | 2014-12-22 | 2015-03-25 | 成都倍特药业有限公司 | Synthesis method for medical-grade deslanoside |
| CN107118248A (en) * | 2017-05-27 | 2017-09-01 | 成都倍特药业有限公司 | A kind of process for purification of high-purity Deslanoside |
| CN109734768A (en) * | 2019-02-01 | 2019-05-10 | 桂林医学院 | Go acetyl cedilanid glucosyl group modified compound composite lipidosome and its application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961326A (en) * | 2012-11-27 | 2013-03-13 | 上海朝晖药业有限公司 | Preparation method of deslanoside injection |
| CN104447933A (en) * | 2014-12-22 | 2015-03-25 | 成都倍特药业有限公司 | Synthesis method for medical-grade deslanoside |
| CN107118248A (en) * | 2017-05-27 | 2017-09-01 | 成都倍特药业有限公司 | A kind of process for purification of high-purity Deslanoside |
| CN109734768A (en) * | 2019-02-01 | 2019-05-10 | 桂林医学院 | Go acetyl cedilanid glucosyl group modified compound composite lipidosome and its application |
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