CN103353498A - High performance liquid chromatography method for determining content of artemisinin in artemisinin extract - Google Patents
High performance liquid chromatography method for determining content of artemisinin in artemisinin extract Download PDFInfo
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- CN103353498A CN103353498A CN2013102381431A CN201310238143A CN103353498A CN 103353498 A CN103353498 A CN 103353498A CN 2013102381431 A CN2013102381431 A CN 2013102381431A CN 201310238143 A CN201310238143 A CN 201310238143A CN 103353498 A CN103353498 A CN 103353498A
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Abstract
The invention discloses a high performance liquid chromatography method for determining the content of artemisinin in artemisinin extract. Specifically, the adopted mobile phase is composed of acetonitrile and water (in a volume ratio of 6:4); the flow rate is 1.0ml/min; the solvent is dissolved by methanol; and the chromatographic column is: C18(4.6*250mm5micrometers); the column temperature is 30DEG C, the sample size is 20 microliters; a UV detector is employed; the detection wavelength is 210nm. Within a sample size of 1-100 micrograms, artemisinin shows a good linear relation with a peak area, and the linear correlation coefficient r is 1. Employment of the high performance liquid method provided in the invention to detect the content of artemisinin in artemisinin extract can shorten the sample analysis time, reduce the detection cost, improve the accuracy and precision of sample analysis, and increase the effective range of sample detection.
Description
Technical field
The present invention relates to a kind of efficient liquid-phase chromatography method of measuring artemislnin content in the qinghaosu medicinal extract.
Background technology
Sweet wormwood is the aerial part of composite family mugwort artemisia annua Artemisia annua L., and qinghaosu is the principal ingredient that is used for the treatment of malaria of extracting in the sweet wormwood.Qinghaosu is the new antimalarial agent of the unique acquisition international recognition of China, artemisinin-based drug treatment pernicious malaria effect is remarkable, become the antimalarial medicine of world health organisation recommendations, the artemisine medicine is not only remarkable to treatment malaria effect, is also obtaining progress aspect the relevant diseases such as antitumor, treatment acquired immune deficiency syndrome (AIDS), kala-azar lupus erythematosus.
Sweet wormwood has another name called artemisia annua, is the sesquiterpene lactone compound (structural formula is seen accompanying drawing 1) that is extract by the feverfew artemisia annua, and molecular formula is C
15H
22O
5, relative molecular mass is 282.33.Extract refers to that medicinal material leaches (or fried) effective ingredient with suitable solvent, boils off part or all of solvent, and is concentrated, adjusts powdery or ointment preparation that concentration is made to required standard.Extract does not contain or contains the minute quantity solvent, and effective constituent is more stable, can store for a long time.How much be divided into extracta spissa and extracta sicca according to water cut, the general water cut of thick medicinal extract is about 15%～20%; The dry extract water cut is about 5%.Medicinal extract contains the ethanol more than 20% at least, if water is the medicinal extract of solvent, its finished product benefit should add 20%～25% ethanol as imitative antiseptic, is beneficial to store.
Except as otherwise herein provided, the every 1ml of extract is equivalent to crude drug 2g～5g.But contain alkaloid or contain the extract of definite effective component extracting, all need through behind the assay, be adjusted to the specification standards of regulation with thinning agent.Thick medicinal extract can use glycerine, liquid glucose to adjust content; Dry extract available starches, lactose, sucrose, magnesium oxide, calcium phosphate, dregs of a decoction fine powder etc. are adjusted content.Except a small amount of extract can be directly used in the clinical practice, as the raw material of other preparations of configuration, extract can be used for disposing tincture, mixture, syrup, tablet, powder, capsule, granule, pill etc. mostly.
Multiple report about the artemislnin content assay method has been arranged at present at home, but the report about artemislnin content assay method in the qinghaosu medicinal extract is also few, therefore explores and improves the efficient liquid-phase chromatography method tool that artemislnin content is measured in the qinghaosu medicinal extract and be of great significance and reference value.
Summary of the invention
The detection method that the purpose of this invention is to provide artemislnin content in the higher qinghaosu medicinal extract of a kind of easy, good separating effect, precision and accuracy.For achieving the above object, this method has adopted following technical scheme:
(1) chromatographic condition: chromatographic column is as filling material take octadecylsilane chemically bonded silica; Mobile phase is the mixed solution of acetonitrile and water, and the volume fraction of its composition is respectively acetonitrile 40～85%, water 15%～60%; Detect wavelength 210nm; 20～45 ℃ of column temperatures; Flow velocity 0.6～1.5ml/min; Sampling volume is 20 μ l, wherein mobile phase preferred peak type and appearance time acetonitrile preferably: water=6: 4 (V/V); Preferred column temperature is 30 ℃, and flow velocity is 1ml/min.
(2) sample preparation: sample adopts ten thousand/balance accurate weighing, and the sample behind the pan paper precision weighing put into the 50ml volumetric flask, add 20ml chromatogram methyl alcohol and ultrasonic 30min dissolving, so that the qinghaosu in the qinghaosu medicinal extract can dissolve fully, and then be settled to scale mark with chromatogram methyl alcohol dilution, shake up the organic filter of rear usefulness 0.2 μ m and filter.
(3) adopt external standard method that the content of qinghaosu in the qinghaosu medicinal extract is measured.
The present invention verifies to method that through tests such as the experiment of precision test, linear relationship, range of linearity experiment and application of sample recovery experiments the result shows the method accurately and reliably, and with class methods relatively, this method detection is faster; Cost is lower; Sensing range is larger; Sensitivity, accuracy are higher.
Description of drawings
Fig. 1 is the structural formula of qinghaosu.
Fig. 2 is that the HPLC of qinghaosu standard specimen detects collection of illustrative plates.
Fig. 3 is that the HPLC of qinghaosu medicinal extract detects collection of illustrative plates.
Fig. 4 is linear relationship chart and the regression equation of qinghaosu.
Embodiment
Following specific embodiment further specifies labor method of the present invention, but the present invention is not limited to the restriction of the scope that claim protects.
Embodiment 1
Chromatographic condition: adopt the Agilent-1260 high performance liquid chromatograph, with C18 reverse-phase chromatographic column (ThermoC
184.6x250mm5 μ m); Mobile phase: acetonitrile: water (6: 4V/V); Flow velocity: 1ml/min; Detect wavelength: 210nm; Detecting device: UV-detector; Sample size: 20 μ l; Acquisition time: 50min; Column temperature: 30 ℃.
Standard items preparations: it is an amount of that precision takes by weighing standard items, places the 50ml volumetric flask, and dilution is settled to scale mark after adding that methyl alcohol is ultrasonic and making sample dissolution, gets solution through 0.2 μ m with the 1ml syringe, disposable organic filter filtration of 13mm.
The testing sample preparation: precision takes by weighing the qinghaosu medicinal extract (the bright Bioisystech Co., Ltd of protecting in Wuhan buys) of about 1g, together place the 50ml volumetric flask with pan paper, adding the ultrasonic 30min of 20ml chromatogram methyl alcohol dissolves sample fully, add methanol constant volume to scale mark and shake up, get solution through 0.2 μ m with the 1ml syringe, disposable organic filter of 13mm filters.
Adopt external standard method, need respectively standard items and testing sample solution to be measured.
According to formula:
The substitution data can obtain artemislnin content.
Standard solution and testing sample are prepared 2 respectively, measure respectively 20 μ l injection liquid chromatographies, and the gained peak area above-mentioned formula of substitution of averaging calculates and get final product.
Measurement result such as table 1:
The high performance liquid chromatography data of table 1 qinghaosu standard specimen and medicinal extract
Show good stability of the present invention by above-mentioned example, degree of accuracy is high, is applicable to the assay of qinghaosu in the qinghaosu medicinal extract.
Methodological study of the present invention
(1) linear relationship experiment
According to the described method configuration of invention qinghaosu standard specimen, volumetric flask configuration concentration with 50ml is the storing solution of 5mg/ml, add the standard solution that methyl alcohol is diluted to respectively 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, 500 μ g/ml, 1000 μ g/ml, 2000 μ g/ml, according to analytical approach of the present invention, sample size (X) and peak area (Y) are carried out data regression (linear relationship chart is seen Fig. 4), regression equation is: Y=44.352X+0.2939, r=1.The result shows that qinghaosu is in 1 μ g～100 μ g concentration ranges, and peak area and sample size are good linear relationship.
(2) precision is investigated
Take by weighing same qinghaosu medicinal extract sample, carry out revision test 6 times, peak area such as table 2:
The peak area of 6 revision tests of table 2 qinghaosu medicinal extract
| Number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area | 5843.9365 | 5837.1231 | 5825.1567 | 5834.7833 | 5837.1983 | 5829.8981 |
Peak area RSD value is 0.45%.The result shows that it is very high that this HPLC method detects artemislnin content precision, and reappearance is fine.
(3) stability test
Get qinghaosu medicinal extract sample, by the inventive method configuration solution, room temperature transfers and sets to 0,4,8,12,24, behind the 48h, according to above-mentioned chromatographic condition, precision is measured 20 μ l injection liquid chromatographies, measures in accordance with the law, calculates the relative standard deviation of qinghaosu peak area, RSD is 0.53% as a result, and the result shows that qinghaosu medicinal extract sample solution is better at the 48h internal stability.
(4) average recovery test
The qinghaosu sample that accurately takes by weighing known content is an amount of, precision adds a certain amount of qinghaosu medicinal extract sample 80%, 100%, 120% respectively again, make in the high, medium and low zone of qinghaosu typical curve each 2 parts respectively of qinghaosu concentration in the need testing solution, by the operation under the item of the present invention, measure in accordance with the law, calculate recovery rate, average recovery rate is 99.89% as a result, RSD is 0.59%, shows that the method average recovery is better.
Claims (6)
1. the method for artemislnin content in the high effective liquid chromatography for measuring qinghaosu medicinal extract is characterized in that chromatographic column is the reverse-phase chromatographic column take octadecylsilane chemically bonded silica as filling material.
2. method according to claim 1 is characterized in that mobile phase is the mixed solution of acetonitrile and water, and wherein the volume fraction of acetonitrile is 40%～85%, and the volume fraction of water is 60%～15%.
3. method according to claim 1 is characterized in that using UV-detector, and the detection wavelength is 210nm, and flow velocity is 0.6～1.5ml/min, 20～45 ℃ of column temperatures; Wherein optimum flow rate is 1ml/min, and column temperature is 30 ℃.
4. method according to claim 1 is characterized in that sample size is 20l.
5. method according to claim 1 is characterized in that the solvent that dissolves qinghaosu and qinghaosu medicinal extract has acetone, ethyl acetate, methenyl choloride, ethanol, methyl alcohol etc., and wherein optimum solvent is methyl alcohol.
6. method according to claim 1 is characterized in that this high performance liquid chromatography adopts external standard method to carry out the assay of qinghaosu; Concentration range is 1 μ g～100 μ g in sample size and the peak area linear relationship.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808827A (en) * | 2014-03-01 | 2014-05-21 | 张家港威胜生物医药有限公司 | High performance liquid chromatography method for measuring content of artesunate |
| CN113189250A (en) * | 2021-03-22 | 2021-07-30 | 禹州市天源生物科技有限公司 | Process quality control technology of single-prescription chicken-used sweet wormwood powder |
Citations (1)
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| CN1904591A (en) * | 2006-08-09 | 2007-01-31 | 重庆医科大学 | Determining method of artemislnin content in artemisia apiacea |
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2013
- 2013-06-17 CN CN2013102381431A patent/CN103353498A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1904591A (en) * | 2006-08-09 | 2007-01-31 | 重庆医科大学 | Determining method of artemislnin content in artemisia apiacea |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808827A (en) * | 2014-03-01 | 2014-05-21 | 张家港威胜生物医药有限公司 | High performance liquid chromatography method for measuring content of artesunate |
| CN113189250A (en) * | 2021-03-22 | 2021-07-30 | 禹州市天源生物科技有限公司 | Process quality control technology of single-prescription chicken-used sweet wormwood powder |
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Application publication date: 20131016 |