CN101339168A - Thunder god vine medicinal materials triptolide content determination method - Google Patents
Thunder god vine medicinal materials triptolide content determination method Download PDFInfo
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- CN101339168A CN101339168A CNA2007100699069A CN200710069906A CN101339168A CN 101339168 A CN101339168 A CN 101339168A CN A2007100699069 A CNA2007100699069 A CN A2007100699069A CN 200710069906 A CN200710069906 A CN 200710069906A CN 101339168 A CN101339168 A CN 101339168A
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Abstract
The invention discloses a test method for the content of triptolide in the crude drugs of common threewingnut root; the test method of the triptolide ever reported has poor precision, low recovery rate and big error and the product quality can not be controlled completely and effectively; the invention adopts a high performance liquid chromatography which comprises the following operation steps: the reference solution is prepared; the triptolide reference is taken and methanol is added so as to prepare solution; the test sample solution is prepared; the common threewingnut root powder is taken and added with methenyl choloride, heating, reflow and cooling are then carried out in sequence; the methenyl choloride is added to make up the lost weight, the obtained solution is shaken to be even and filtered; subsequent filtrate is taken and the solvent is dried; the residue is added with the methenyl choloride for fractional dissolution; the obtained mixture is arranged on a processed aluminium oxide column, and is eluted by using ethyl acetate-absolute ethyl alcohol; the elution is collected and evaporated to dryness; the residue is added with the methanol for dissolution, shaken to be even and filtered, and the subsequent filtrate is taken; the reference solution and the test sample solution are taken respectively to be injected into a liquid chromatograph. The invention can control the quality of the crude drugs of the common threewingnut root effectively and ensures the good material of the finished product by the common threewingnut root.
Description
Technical field
The present invention relates to the assay of material, the content assaying method of triptolide in specifically a kind of tripterygium wilfordii.
Background technology
Thunder godvine (Tripterygium Wilfordii Hook.f.) another name gelsemium elegan, herba fibraureae recisae, cabbageworm medicine, for defending the Mao Ke tripterygium plant, fallen leaves rattan shape shrub, it has multiple pharmacological effect such as anti-inflammatory, antibiotic, immunological regulation, antitumor and antifertility, is first-selected both at home and abroad immunodepressant.A large amount of pharmacology and clinical researches show that triptolide (triptolide) is the topmost active component of thunder godvine, also are main toxic components.Tripterygium wilfordii mainly picks up from wild at present, quality is uneven, main active such as triptolide are very unstable, triptolide massfraction mxm. and minimum differ 50 times between Different Individual, different groups triptolide massfraction mxm. and minimum differ 10 times, have a strong impact on the drug safety and the curative effect of thunder godvine preparation.Realize thunder godvine preparation " safety, effective, stable, controlled ", must be from source control tripterygium wilfordii quality.Therefore for guaranteeing the validity and the security of the clinical application of thunder godvine preparation, the method for quality control of setting up an effective tripterygium wilfordii seems particularly important.In the present relevant document both at home and abroad, the assay method of triptolide once had report in the tripterygium wilfordii, but poor through its precision of verification experimental verification, the recovery is low, and error is big, can not control product quality completely effectively.
Summary of the invention
Technical matters to be solved by this invention is to overcome the defective that above-mentioned prior art exists, the content assaying method of the triptolide that a kind of specificity is strong, precision good, the recovery is high, measurement result is correct is provided, can effectively control the quality of tripterygium wilfordii, to guarantee the quality raw materials of thunder godvine manufactured goods.
For this reason, the present invention adopts following technical scheme: the content assaying method of triptolide in the tripterygium wilfordii, adopt high performance liquid chromatography, and its operation steps is as follows: the 1) preparation of reference substance solution: get the triptolide reference substance and add methyl alcohol and make solution; 2) preparation of need testing solution: get tripterygium wilfordii powder (crossing sieve No. two) and add methenyl choloride, reflux is put cold, add methenyl choloride and supply the weight that subtracts mistake, shake up, filter, get subsequent filtrate and volatilize solvent, residue adds methenyl choloride gradation dissolving, places on the alumina column of having handled well, with ethyl acetate-absolute ethyl alcohol wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol makes its dissolving, shakes up, and filters, get subsequent filtrate, promptly; 3) get reference substance solution and need testing solution respectively and inject liquid chromatograph, with the octadecylsilane chemically bonded silica is filling agent, the mixed liquor of sodium dihydrogen phosphate-acetonitrile-methyl alcohol is a moving phase, and the detection wavelength is 218nm, calculates the content of triptolide with external standard method.
The content assaying method of described triptolide, change its adsorption activity by the water cut of regulating aluminium oxide, the optimum moisture content of adsorbent aluminium oxide is 8%, the adsorption activity of this moment is the strongest, its preparation method is as follows: get 105 ℃ of bakings of aluminium oxide 3 hours, put to warm, dripping purified water to water cut by weight is 8%, shake up when dripping, promptly.
The content assaying method of described triptolide, the concentration of used sodium dihydrogen phosphate are 0.05mol/L, transfer pH value to 4.0 with phosphoric acid, and sodium dihydrogen phosphate: acetonitrile: the volume ratio of methyl alcohol is 60: 15: 25.
The content assaying method of described triptolide, the volume ratio of ethyl acetate and absolute ethyl alcohol are 1: 1.
The content assaying method of described triptolide, the quality percentage composition of triptolide answers 〉=0.001% in the tripterygium wilfordii.
The method specificity that the present invention adopts is strong, precision good, the recovery is high, measurement result is correct, can effectively control the quality of tripterygium wilfordii, guaranteed the quality raw materials of thunder godvine manufactured goods, guaranteed the drug safety and the curative effect of thunder godvine preparation.
The present invention is further illustrated below in conjunction with embodiment, but the present invention is not limited to these embodiment.
Embodiment
[assay]
Used instrument:
Tianjin, island high performance liquid chromatograph, LC-10ATvp
Tianjin, island ultraviolet multiwavelength detector, SPD-10Avp
F door C18 post, 5 μ m, 250mm * 4.6mm
Detect wavelength: 218nm.
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filling agent; With the sodium dihydrogen phosphate of 0.05mol/L (transferring pH value to 4.0 with phosphoric acid)-acetonitrile-methyl alcohol=60: 15: 25 be moving phase, treated that triptolide went out behind the peak about two minutes, adopted gradient elution, usefulness methanol-eluted fractions 10 minutes, and flow velocity is 1ml/min; The detection wavelength is 218nm; Number of theoretical plate calculates by the triptolide peak should be not less than 3000.
The preparation of reference substance solution: it is an amount of that precision takes by weighing the triptolide reference substance, adds methyl alcohol and make the solution that every 1ml contains 10 μ g, promptly.
The preparation of need testing solution: get the about 5.0g of this product powder (crossing sieve No. two), the accurate title, decide, and puts in the tool round-bottomed flask, the accurate methenyl choloride 50ml that adds claims to decide weight, reflux 3 hours, put cold, add methenyl choloride and supply the weight that subtracts mistake, shake up, filter, precision is measured subsequent filtrate 25ml, volatilize solvent, residue adds 5ml methenyl choloride gradation dissolving, puts (the about 0.9cm of internal diameter on the alumina column of having handled well, 100～200 orders contain 8% moisture neutral alumina 2g, 20ml methenyl choloride dress post, prewashing), with ethyl acetate-absolute ethyl alcohol (1: 1) 30ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol makes dissolving, is transferred in the 10ml measuring bottle, adds methyl alcohol to scale, shake up, filter, get subsequent filtrate, promptly.
Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph and measure.
Linear test
It is an amount of to get the triptolide reference substance, add methyl alcohol and make the solution that contains triptolide 1.416 μ g/ml, 2.832 μ g/ml, 5.664 μ g/ml, 11.328 μ g/ml, 22.656g/ml respectively, according to chromatographic condition under [assay] item, the accurate respectively 20 μ l of absorption inject liquid chromatograph, measure the peak area of triptolide, with the peak area is ordinate, is that horizontal ordinate carries out linear regression with concentration, sees Table 1.
The linear regression of table 1 triptolide
Triptolide is the good linear relation in 1.416 μ g/ml～22.656 μ g/ml concentration ranges as a result.
The specificity test
Get this product assay agents useful for same and lack tripterygium root, root prepares negative need testing solution according to [assay] method, measures according to [assay] method.
The result: negative test sample is noiseless to this product assay.
Detectability
Solution stepwise dilution under the line taking test item, signal to noise ratio (S/N ratio) are that detection in 3: 1 o'clock is limited to 2.832ng.
Quantitative limit
Solution stepwise dilution under the line taking test item, signal to noise ratio (S/N ratio) are that detection in 10: 1 o'clock is limited to 9.44ng.
The test of returning property rate
Get the about 2.0g of tripterygium root sample, put in the tool plug conical flask, add triptolide reference substance solution (methenyl choloride preparation) 4.00ml of 14.7 μ g/ml respectively, add methenyl choloride 46.00ml, prepare need testing solution according to [assay] method;
Get the about 2.5g of tripterygium root sample, put in the tool plug conical flask, add triptolide reference substance solution (methenyl choloride preparation) 5.00ml of 14.7 μ g/ml respectively, add methenyl choloride 45.00ml, prepare need testing solution according to [assay] method;
Get the about 3.0g of tripterygium root sample, put in the tool plug conical flask, add triptolide reference substance solution (methenyl choloride preparation) 6.00ml of 14.7 μ g/ml respectively, add methenyl choloride 44.00ml, prepare need testing solution according to [assay] method;
Get triptolide reference substance 0.0125g, add the solution that methyl alcohol is made 10 μ g/ml, be reference substance solution.
Get need testing solution and reference substance solution respectively and measure, the results are shown in Table 2 according to [assay] method.
Table 2 triptolide recovery test result
The result: measure triptolide content with this law, the recovery is good, measures accurately.
Solution stability testing
Get the about 5.0g of tripterygium root sample, the accurate methenyl choloride 50ml that adds prepares need testing solution according to [assay] method, measures at 0,1,2,4,8,24 hour respectively, the results are shown in Table 3.
Table 3 need testing solution stability test result
The result: this product need testing solution is stable in 24 hours.
Precision test (replica test)
Get the about 5.0g of tripterygium root sample, 6 parts, the accurate methenyl choloride 50ml that adds prepares need testing solution according to [assay] method;
Get triptolide reference substance 0.0125g, add the solution that methyl alcohol is made 10 μ g/ml, be reference substance solution.
Get need testing solution and reference substance solution respectively and measure, the results are shown in Table 4 according to [assay] method.
Table 4 replica test result
The result: measure triptolide content with this law, repeatability is good, the precision height.
Precision test (middle precision test)
Get the about 5.0g of tripterygium root sample, 6 parts, the accurate methenyl choloride 50ml that adds prepares need testing solution according to [assay] method;
Get triptolide reference substance 0.0125g, add the solution that methyl alcohol is made 10 μ g/ml, be reference substance solution.
Get need testing solution and reference substance solution respectively and measure, the results are shown in Table 5 according to [assay] method.
Precision test result in the middle of the table 5
The result: measure triptolide content with this law, middle precision is good.
Claims (6)
1, the content assaying method of triptolide in the tripterygium wilfordii adopts high performance liquid chromatography, and its operation steps is as follows: the 1) preparation of reference substance solution: get the triptolide reference substance and add methyl alcohol and make solution; 2) preparation of need testing solution: get the tripterygium wilfordii powder and add methenyl choloride, reflux is put cold, add methenyl choloride and supply the weight that subtracts mistake, shake up, filter, get subsequent filtrate and volatilize solvent, residue adds methenyl choloride gradation dissolving, places on the alumina column of having handled well, with ethyl acetate-absolute ethyl alcohol wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol makes its dissolving, shakes up, and filters, get subsequent filtrate, promptly; 3) get reference substance solution and need testing solution respectively and inject liquid chromatograph, with the octadecylsilane chemically bonded silica is filling agent, the mixed liquor of sodium dihydrogen phosphate-acetonitrile-methyl alcohol is a moving phase, and the detection wavelength is 218nm, calculates the content of triptolide with external standard method.
2, the content assaying method of triptolide according to claim 1 is characterized in that changing its adsorption activity by the water cut of regulating aluminium oxide.
3, the content assaying method of triptolide according to claim 2, the water cut that it is characterized in that alumina adsorbant is 8%.
4, the content assaying method of triptolide according to claim 1 and 2, the concentration that it is characterized in that used sodium dihydrogen phosphate is 0.05mol/L, transfer pH value to 4.0 with phosphoric acid, the volume ratio of sodium dihydrogen phosphate-acetonitrile-methyl alcohol is 60: 15: 25.
5, the content assaying method of triptolide according to claim 4, the volume ratio that it is characterized in that ethyl acetate and absolute ethyl alcohol is 1: 1.
6, the content assaying method of triptolide according to claim 5 is characterized in that the quality percentage composition of triptolide in the tripterygium wilfordii answers 〉=0.001%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033570A (en) * | 2011-09-29 | 2013-04-10 | 广州陈李济药厂有限公司 | Quality detection method for tripterygii hypoglauci medicinal materials |
| CN105758948A (en) * | 2016-02-25 | 2016-07-13 | 中国农业科学院蜜蜂研究所 | Application of triptolide in identification presence of tripterygium glycoside honey in honey as feature identification material |
| CN106153794A (en) * | 2015-04-10 | 2016-11-23 | 浙江海洋学院 | The method of triptolide in a kind of microwave radiation exaraction/HPLC detection tripterygium leaf |
| CN109239213A (en) * | 2018-09-13 | 2019-01-18 | 重庆市药研院制药有限公司 | A kind of detection method of colguhoumia root preparation triptolide |
-
2007
- 2007-07-03 CN CNA2007100699069A patent/CN101339168A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033570A (en) * | 2011-09-29 | 2013-04-10 | 广州陈李济药厂有限公司 | Quality detection method for tripterygii hypoglauci medicinal materials |
| CN103033570B (en) * | 2011-09-29 | 2014-08-20 | 广州白云山陈李济药厂有限公司 | Quality detection method for tripterygii hypoglauci medicinal materials |
| CN106153794A (en) * | 2015-04-10 | 2016-11-23 | 浙江海洋学院 | The method of triptolide in a kind of microwave radiation exaraction/HPLC detection tripterygium leaf |
| CN105758948A (en) * | 2016-02-25 | 2016-07-13 | 中国农业科学院蜜蜂研究所 | Application of triptolide in identification presence of tripterygium glycoside honey in honey as feature identification material |
| CN105758948B (en) * | 2016-02-25 | 2018-02-27 | 中国农业科学院蜜蜂研究所 | Whether triptolide contains the application in tripterygium wilfordii honey as feature recognition thing in honey is differentiated |
| CN109239213A (en) * | 2018-09-13 | 2019-01-18 | 重庆市药研院制药有限公司 | A kind of detection method of colguhoumia root preparation triptolide |
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Open date: 20090107 |