CN101504396A - Method for detecting ecdysterone in pharmaceutical composition - Google Patents
Method for detecting ecdysterone in pharmaceutical composition Download PDFInfo
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- CN101504396A CN101504396A CNA2009101188873A CN200910118887A CN101504396A CN 101504396 A CN101504396 A CN 101504396A CN A2009101188873 A CNA2009101188873 A CN A2009101188873A CN 200910118887 A CN200910118887 A CN 200910118887A CN 101504396 A CN101504396 A CN 101504396A
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Abstract
A method for detecting the content of ecdysterone in an ecdysterone-containing pharmaceutical composition is characterized in that a chromatographic column is an octadecylsilane chemically bonded silica packed column, and an ultraviolet-visible light detector is adopted to detect the analysis result, wherein the detection wavelength is 240-255 nm, the mobile phase can be acetonitrile-water, and the column temperature is 25-35 ℃. The present invention also provides a method for controlling the quality of a product by tracking the concentration of ecdysterone in an extract solution using high performance liquid chromatography in the decocting or extracting step of a medicinal material, wherein the decocting or extracting operation is terminated after a target value is reached. The method disclosed by the invention is simple to operate, strong in specificity, good in repeatability, high in accuracy of content measurement results, and capable of better controlling the quality of the product.
Description
Technical field
The present invention relates to a kind of method that is used for detection of drugs composition ecdysterone.
Background technology
Ecdysterone claims β-decortication pine again, and β-moulting hormone has effects such as blood sugar lowering and cholesterol, anti-arrhythmia, antiviral, anti-inflammatory, anti-oxidant and immunological enhancement.Natural being present in the amaranthaceous plant root of bidentate achyranthes plants such as (Achyranthes bidentata Bl.).A lot of Chinese medicine preparations are active component with it.For example, a kind of promptly is monarch drug in a prescription with the achyranthes aspera by the refining pharmaceutical composition fully of achyranthes aspera, Malan Straw, Asiatic plantain and day name, said composition can be clearing heat and detoxicating, relieving sore throat and acesodyne, be used for that pharyngeal due to the lung sthenia gastropyrexia swollen and ache, heating, thirsty, constipation, and the treatment or the alleviation of all diseases such as acute tonsillitis, acpuei pharyngitis.
For the pharmaceutical composition that contains ecdysterone, especially above-mentioned by the refining pharmaceutical composition that is equipped with of achyranthes aspera, Malan Straw, Asiatic plantain and day name, the method for quality control that has earlier is to adopt the oleanolic acid content after acid hydrolysis in the tlc-scanning determination finished product.Its concrete operations are as follows:
Get above-mentioned by achyranthes aspera, Malan Straw, smart and the Asiatic plantain pharmaceutical compositions 25ml of it name puts in the separating funnel, the water spore and normal butyl alcohol extraction 6 times, each 20ml merges normal butyl alcohol liquid, puts evaporate to dryness in the water-bath, residue adds ethanol 30ml, hydrochloric acid 3ml makes dissolving, puts in the water-bath heating and refluxing extraction 1 hour, and extract reclaims ethanol to there not being the alcohol flavor, add water 30ml, be transferred in the separating funnel, extract 6 times, each 20ml with sherwood oil (60～90 ℃) jolting, merge sherwood oil liquid, put evaporate to dryness in the water-bath, residue adds the absolute ethyl alcohol low-grade fever makes dissolving, quantitatively is transferred in the 5ml measuring bottle, cooling, be diluted to scale with absolute ethyl alcohol, shake up, as need testing solution, other evens up pier tartaric acid reference substance, the accurate title, decide, and adds absolute ethyl alcohol and make the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw above-mentioned need testing solution 6 μ l, reference substance solution 2 μ l and 4 μ l, the point of crossing is on same silica gel g thin-layer plate respectively, with cyclohexane-methenyl choloride-ethyl acetate (20: 5: 8) is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃, takes out, on thin layer plate, cover onesize glass plate, use immobilization with adhesive tape on every side, scan wavelength X s=520nm according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B thin layer chromatography scanning), λ R=700nm, measure test sample absorbance integrated value and reference substance absorbance integrated value, calculate, promptly.
This method complex operation, poor reproducibility, assay result's error is big, is difficult to control the quality of finished product.
Summary of the invention
The purpose of this invention is to provide a kind of method of analyzing ecdysterone content in the pharmaceutical composition simply, reliably.
Another object of the present invention provides a kind of method for quality of controlling a kind of by achyranthes aspera, Malan Straw, Asiatic plantain and day refining pharmaceutical composition that is equipped with of name simply, reliably.
An aspect of of the present present invention relates to a kind of method that is used for detecting the pharmaceutical composition ecdysterone content that contains ecdysterone, this detection is undertaken by high performance liquid chromatography, wherein chromatographic column is the octadecylsilane chemically bonded silica packed column, adopt ultraviolet-visible detector check and analysis result, detect wavelength and be 240 to 255nm, moving phase can be acetonitrile-water, and column temperature is 25 to 35 ℃.In a kind of exemplary embodiment, chromatographic column is Aglient TC-C
18, the detection wavelength is 248nm, and moving phase is 15: 85 acetonitrile-water, and column temperature is 30 ℃.
The invention still further relates to a kind of drug quality control method of controlling ecdysterone content in the pharmaceutical composition, it is by in the decoction of medicinal material or extract link and follow the tracks of ecdysterone concentration in the extract with high performance liquid chromatography, after reaching a desired value, stop decocting or extracting, wherein, testing conditions is: chromatographic column is the octadecylsilane chemically bonded silica packed column, adopt ultraviolet-visible detector check and analysis result, detect wavelength and be 240 to 255nm, moving phase can be acetonitrile-water, and column temperature is 25 to 35 ℃.Preferably, chromatographic column is AglientTC-C
18, the detection wavelength is 248nm, and moving phase is 15: 85 acetonitrile-water, and column temperature is 30 ℃.
In a kind of typical embodiment, described pharmaceutical composition is fully refining by achyranthes aspera, Malan Straw, Asiatic plantain and day name.
Aforementioned pharmaceutical compositions can be made by following steps, the decocting that the four traditional Chinese medicine material is added 18-30 times of weight boils, keep the decoction liquor constant volume in the decoction process, detect the concentration of ecdysterone in the decoction liquor with high performance liquid chromatography, when ecdysterone content reaches 0.01mg/ml in detecting sample, stop to decoct or extracting.
In said method, be used to extract 20 to 25 times of amounts that the used water yield of medicine preferably maintains medicinal material, most preferably in 20 times of amounts (by weight) of medicinal material.
This content assaying method is simple to operate, strong, the good reproducibility of specificity, and assay result precision height can better be controlled the quality of product.
Description of drawings
Fig. 1 is the canonical plotting according to the inventive method obtained.
Embodiment
In the method for the invention, adopt the content of the ecdysterone in the high performance liquid chromatography detection of drugs composition.Chromatographic column is the octadecylsilane chemically bonded silica packed column, preferred Shim-Pack CLC-ODS chromatographic column, Hypersil-ODS chromatographic column and Aglient TC-C
18, Aglient TC-C most preferably
18Adopt ultraviolet-visible detector check and analysis result, detect wavelength and be 240, most preferably be 248nm to 255nm; Moving phase can be arbitrary proportioning of methyl alcohol-water, acetonitrile-water, is preferably acetonitrile-water (15: 85); Column temperature is 25 to 35 ℃, is preferably 30 ℃.
In order to detect, specimen need be mixed with solution.For this reason, often diluted sample is become to subscribe the methyl alcohol or the ethanolic solution of concentration, get subsequent filtrate then and detect.
In a kind of exemplary embodiment of the present invention, adopt following condition to carry out the content detection of ecdysterone:
Instrument: Waters 1525 high performance liquid chromatographs
Detecting device: Waters 2996
Detect wavelength 248nm
Moving phase: acetonitrile-water (15: 85)
Chromatographic column: Aglient TC-C
18(250mm * 4.6mm * 5 μ m)
Column temperature: 30 ℃ of chromatographic work station: Empower
The ecdysterone reference substance: (lot number: 111638-200603 Nat'l Pharmaceutical ﹠ Biological Products Control Institute, use for assay).
Below with a kind of be example by achyranthes aspera, Malan Straw, Asiatic plantain and the refining pharmaceutical composition that is equipped with of day name, describe the present invention in detail.
A kind of typical preparation method of said composition is as follows:
More than four flavor boilings twice, each 1.5 hours, add for the first time 14 times of water gagings, add 12 times of water gagings for the second time, collecting decoction filters, filtrate is concentrated into the clear cream that relative density is 1.07～1.09 (60 ℃).Get sucrose 250g and make syrup, merge, stir evenly with above-mentioned clear cream, cooling, standby; Other gets menthol 0.2g, Sodium Benzoate 3g, and essence is an amount of, behind 70% dissolve with ethanol, is added in the above-mentioned soup, stirs evenly, and adjusts total amount to 1000ml, stirs evenly, and leaves standstill, and filters, promptly.
Need testing solution preparation method's selection
Precision is measured composition sample 25ml (2 parts), puts in the 50ml volumetric flask, adds methyl alcohol, ethanol dilution respectively to scale, shakes up, and filters, and gets subsequent filtrate, promptly.
Accurate each the 5 μ l of reference substance solution and need testing solution that draw of determination method inject liquid chromatograph, measure also and calculate, and employing methyl alcohol, ethanol are as the measurement result no significant difference (seeing Table 1) of diluting solvent.Because of the toxicity of ethanol less, so select ethanol as solvent.Method is: precision is measured sample 25ml, puts in the 50ml measuring bottle, adds ethanol dilution to scale, shakes up, and filters, and gets subsequent filtrate, promptly gets need testing solution.
Table 1 need testing solution preparation method's selection experimental result
The mensuration of negative control solution
An amount of by all the other each medicinal materials that prescription takes by weighing except that achyranthes aspera, make the negative sample that lacks achyranthes aspera by preparation technology, with the negative sample solution of the scarce achyranthes aspera of method preparation, measure in accordance with the law.
Result: in the chromatogram of negative control solution,, illustrate that negative control solution is noiseless at the colourless spectrum peak that exists together mutually of the retention time with ecdysterone reference substance chromatographic peak.
Linear relationship is investigated
Accurate ecdysterone reference substance solution (0.054mg/ml) 1,3,5,7,9, the 15 μ l that draw inject liquid chromatograph, measure its peak area in accordance with the law.Sample size with ecdysterone is a horizontal ordinate, and peak area is an ordinate, drawing standard curve (seeing Table 2).Its regression equation is: y=1282.3x+7831.1, R=0.9998.The result shows that the sample size of ecdysterone concerns good (see figure 1) in 54ng～810ng scope internal linear.
Table 2 standard curve determination result
The precision test
The same need testing solution of accurate absorption (lot number: 20060501), sample introduction 6 times, each 5 μ l measure, and the RSD of its ecdysterone peak area integrated value is 2.1%, and the result shows precision good (seeing Table 3).
Table 3 Precision test result table
Stability test
Get that same (lot number: 20060501) need testing solution, respectively at 0,2,4,7,13,20,25,35 hour after the preparation, the accurate 5 μ l that draw injected liquid chromatograph, measure, and the results are shown in Table 4.
Table 4 stability test is table as a result
The result shows: need testing solution is basicly stable in back 35 hours of preparation.
Replica test
(lot number: 20060501) the test sample sample is 6 parts, according to measuring with quadrat method, the results are shown in Table 5 to get same lot number.
Table 5 replica test result
The result shows: this method repeatability better.
Recovery test
Get
MeasureThe same lot number of content (lot number: 20060501) sample (containing ecdysterone 0.1283mg/ml) 3ml, 15ml, 25ml, put in the 50ml measuring bottle, accurate respectively reference substance solution (0.13484mg/ml) 3ml, 10ml, the 20ml of adding adds 50% ethanol dilution to scale and mensuration.The results are shown in Table 6.
Table 6 recovery test is table as a result
The result shows: this method average recovery is better.Wherein numbering 1-6 and 13-18 is scope checking result, and average recovery rate is 102.2%, and RSD is 1.57%.
Above-mentioned result of study shows that this content assaying method is simple to operate, and specificity is strong, good reproducibility, assay result precision height.
Another aspect of the present invention provides a kind of drug quality control method by ecdysterone content in the high performance liquid chromatography control pharmaceutical composition.It passes through in the decoction of medicinal material or extracts link with the ecdysterone concentration in the high performance liquid chromatography tracking extract, after reaching a desired value, stops decocting or extraction.
In the specific embodiment aspect above-mentioned, be used for controlling the ecdysterone content that adopts the clear oral liquid of laryngopharynx of aforementioned four flavor Chinese crude drugs productions by applicant company.
In order to adopt said method, the inventor improves existing processes, promptly, no longer adopt the extraction method that repeatedly repeats of the prior art, but add the water of capacity once or other extract solvent, for example ethanol extracts continuously or decocts, the concentration of the ecdysterone in decoction process in the Detection and Extraction liquid.When reaching a predetermined value, stop to decoct or extracting operation.Adopt this method, simplified technology, the constant product quality that makes,
The inventor finds, adopts method of the present invention, and the amount that is used to extract the solvent of medicine is a key factor that influences process efficiency and product quality.The dosage of solvent can not be too small, if too small, even the time of extracting is extended, the active component in the medicinal material can not effectively be separated out; The amount of solvent also is unsuitable for excessive, if excessive, the burden of the corresponding increase subsequent job of meeting.The inventor finds that for the situation that water is extracted, under situation about decocting, the utilization of water can not be lower than 18 times of amounts of medicinal material by weight.But be not higher than 30 times of amounts usually.For the situation that adopts ethanol, use amount should be between 24 to 35 times of amounts.Most preferred condition is to adopt the water of 20 to 25 times of amounts, the water of preferred especially 20 times of amounts.In all cases, in leaching process, should keep the solvent of constant.
The clear oral liquid of producing with applicant company of a kind of laryngopharynx is a specimen, and this product is also made by aforementioned four flavor Chinese crude drugs.The assay method of operating is as follows:
Chromatographic condition: chromatographic column is Aglient TC-C
18(250mm * 4.6mm * 5 μ m); Moving phase is acetonitrile-water (15: 85); The detection wavelength is 248nm; 30 ℃ of column temperatures.Number of theoretical plate calculates by the ecdysterone peak and is not less than 2000.
The preparation of reference substance solution: it is an amount of that precision takes by weighing the ecdysterone reference substance, adds Diluted Alcohol and make the solution that every 1ml contains 30 μ g, promptly.
The preparation of need testing solution: get the clear oral liquid sample of laryngopharynx, mixing, precision is measured 25ml, puts in the 50ml measuring bottle, adds ethanol dilution to scale, shakes up, and filters, and gets subsequent filtrate, promptly.
Determination method: accurate respectively reference substance solution and each 5 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly.
According to content assaying method provided by the invention, measured the content of 15 batches of clear oral liquid samples of laryngopharynx, every batch sample replicate determination twice the results are shown in Table 7.
The clear oral liquid sample size of table 7 laryngopharynx measurement result
Embodiment 2
According to the identical method of embodiment 1, measured the content of 10 batches of achyranthes aspera medicinal materials, every batch sample replicate determination twice the results are shown in Table 8.
Ten batches of achyranthes aspera medicinal materials of table 8 assay result
Press recipe quantity and calculate, prepare the clear oral liquid of every 1ml laryngopharynx and need achyranthes aspera 0.25g.After measured, the average content of 10 batches of achyranthes aspera medicinal materials is 0.04938% with percentage by weight.Through surveying 15 batches of clear oral liquid samples of laryngopharynx, average content is 0.08383mg/ml, and the rate of transform is 67.9%.
Through surveying 15 batch samples, average content is 0.08383mg/ml, by 70% conversion, is 0.058mg/ml, so tentative content limit is: every 1ml contains achyranthes aspera in ecdysterone (C27H34O11), must not be less than 50 μ g.
Above-mentioned test findings shows: this method repeatability is better, assay accuracy height, and error is little.
Quality monitoring in the clear oral liquid production run of embodiment 3 laryngopharynx
Four flavor formula ratio medicinal materials add 20 times of water gagings and decoct, and keep the decoction liquor constant volume in the decoction process.Detect the concentration of ecdysterone in the decoction liquor with high performance liquid chromatography.Testing conditions and operation wherein, are got decoction liquor with embodiment 1, filter just, and precision is measured 25ml, puts in the 50ml measuring bottle, adds ethanol dilution to scale, shakes up, and filters, and gets subsequent filtrate, sample detection.When ecdysterone content reaches 0.01mg/ml in detecting sample, stop to decoct.Every BT(batch testing) product carry out twice, and in Kong Zhi the product, the content of ecdysterone reaches 0.075mg/ml by this method.Under the condition of present embodiment, only need promptly finish extraction step in 1 to 3 hour usually.Technology saves time most, and product quality is very stable.The stable content of ecdysterone is more than 0.05mg/ml in the product.
Claims (8)
1. method that is used for detecting the pharmaceutical composition ecdysterone content that contains ecdysterone, it is characterized in that, this detection is undertaken by high performance liquid chromatography, chromatographic column is the octadecylsilane chemically bonded silica packed column, adopt ultraviolet-visible detector check and analysis result, detect wavelength and be 240 to 255nm, moving phase can be acetonitrile-water, and column temperature is 25 to 35 ℃.
2. method according to claim 1, wherein chromatographic column is Aglient TC-C
18, the detection wavelength is 248nm, and moving phase is 15: 85 acetonitrile-water, and column temperature is 30 ℃.
3. drug quality control method of controlling ecdysterone content in the pharmaceutical composition, it is by in the decoction of medicinal material or extract link and follow the tracks of ecdysterone concentration in the extract with high performance liquid chromatography, after reaching a desired value, stop decocting or extracting, wherein, testing conditions is: chromatographic column is the octadecylsilane chemically bonded silica packed column, adopt ultraviolet-visible detector check and analysis result, detect wavelength and be 240 to 255nm, moving phase can be acetonitrile-water, and column temperature is 25 to 35 ℃.
4. method according to claim 3, wherein chromatographic column is Aglient TC-C
18, the detection wavelength is 248nm, and moving phase is 15: 85 acetonitrile-water, and column temperature is 30 ℃.
5. according to claim 3 or 4 described methods, wherein, described pharmaceutical composition is fully refining by achyranthes aspera, Malan Straw, Asiatic plantain and day name.
6. method according to claim 5, may further comprise the steps: the decocting that the four traditional Chinese medicine material is added 18-30 times of weight boils, keep the decoction liquor constant volume in the decoction process, detect the concentration of ecdysterone in the decoction liquor with high performance liquid chromatography, when ecdysterone content reaches 0.01mg/ml in detecting sample, stop to decoct or extracting.
7. method according to claim 6, wherein, the used water yield maintains 20 to 25 times of amounts of medicinal material.
8. method according to claim 6, wherein, the used water yield maintains 20 times of amounts of medicinal material.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837615A (en) * | 2014-02-15 | 2014-06-04 | 青岛农业大学 | Method applying HPLC and simultaneously determining phytoecdysone substance |
| CN108459100A (en) * | 2018-03-21 | 2018-08-28 | 四川省中药饮片有限责任公司 | A kind of detection method of radix cyathulae medicinal material |
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2009
- 2009-03-05 CN CNA2009101188873A patent/CN101504396A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837615A (en) * | 2014-02-15 | 2014-06-04 | 青岛农业大学 | Method applying HPLC and simultaneously determining phytoecdysone substance |
| CN103837615B (en) * | 2014-02-15 | 2015-05-20 | 青岛农业大学 | Method applying HPLC and simultaneously determining phytoecdysone substance |
| CN108459100A (en) * | 2018-03-21 | 2018-08-28 | 四川省中药饮片有限责任公司 | A kind of detection method of radix cyathulae medicinal material |
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Application publication date: 20090812 |