CN101357204A - Quality control method of traditional Chinese medicine preparation for treating pain - Google Patents

Abstract

The invention discloses a quality control method of traditional Chinese medicine preparation, which is a quality control method of hurt resisting capsule for treating pain. The quality control method achieves the aim of effectively controlling the quality of products by distinguishing or measuring the content of one or a plurality of dandelion, east Asian tree fern rhizome and red paeonia in the prescription. The details are shown in attached diagrams. The quality control method has the advantages that by the identification and content measurement of the multiple raw material medicines in the prescription, the drug effect and stability of the produced hurt resisting capsule can be effectively ensured, thus better improving the safety of using the medicine by patients.

Description

A kind of method of quality control for the treatment of grieved Chinese medicine preparation

Technical field

The present invention relates to a kind of method of quality control of Chinese medicine preparation, relate in particular to a kind of method of quality control for the treatment of grieved Chinese medicine preparation.

Background technology

Preparation SHANGTONGNING JIAONANG of the present invention has blood circulation promoting and blood stasis dispelling, reducing swelling and alleviating pain; Accelerate growth of spur; Promote fracture heals in early days; Promoting the effect of wound surface injury repairing, is for the very effective a kind of Chinese medicine preparation of the treatment of bone injury.In the existing kind, there is 'Shangtongning ', but, guarantees that the drug safety of extensive patients but rarely has the people to study for quality how better to control it with the kind different dosage form.The present invention is exactly in order can better to control the quality of this product, to reach the therapeutic effect of better and safer.

Data shows, takes the Chinese medicine time of containing Aristolochic Acid continuously the longlyest can not surpass one month, otherwise can cause the retention toxicosis reaction, and this poisoning is invisible, directly damages the human kidney organ, and severe patient may be dead because of renal failure.

Cause the report of kidney damage to start from 1993 about Aristolochic Acid, have the medium of states such as Belgium, Britain, Poland, France, Japan to disclose in succession because of taking the case that the treatment by Chinese herbs eczema that contains Aristolochic Acid or fat-reducing cause renal dysfunction.Through looking into, the arch-criminal is exactly the Aristolochic Acid in the medical materials such as Caulis Aristolochiae Manshuriensis and Radix Aristolochiae Fangchi.

So, in order to guarantee not contain Aristolochic Acid in the raw materials used medicine Herba Asari in the preparation of the present invention, we check Herba Asari by the method for assay, find not detect Aristolochic Acid, thereby can guarantee the drug safety of the quality and the extensive patients of medicine better.

Summary of the invention

The purpose of this invention is to provide a kind of method of quality control for the treatment of grieved Chinese medicine preparation, it is the method for quality control by the prepared SHANGTONGNING JIAONANG of crude drug Olibanum (processed), Myrrha (processed), Radix Et Rhizoma Nardostachyos, Rhizoma Corydalis (processed with vinegar), Herba Asari, Rhizoma Cyperi (processed), Rhizoma Kaempferiae, the Radix Angelicae Dahuricae, better guarantee the stability of medicine with this, quality controllability makes medication safer, effective.

The objective of the invention is to realize by the following technical solutions, comprise the determination limit of Aristolochic Acid in following discriminating and/or microscopical identification and/or the Herba Asari:

The discriminating of Rhizoma Corydalis

Get this product content 1.5-4.5g, the 10-30ml that adds diethyl ether flooded 0.5-1.5 hour, and ether solution inclines, add strong ammonia solution 1.5-4.5ml and make moisteningly, the 10-30ml that adds diethyl ether dipping 1.5-4.5 hour filters, filtrate evaporate to dryness, residue add dehydrated alcohol 0.5-1.5ml makes dissolving, as need testing solution.Other gets the tetrahydropalmatine reference substance, adds dehydrated alcohol and makes the solution that contains 0.05-0.15mg among every 0.5-1.5ml, in contrast product solution.According to the thin layer chromatography test, draw each 2.5-7.5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene-acetone (8-25: 1.5-4.5) be developing solvent, launch, take out, dry, it is smoked clear to the speckle colour developing to put in the iodine steam, fling to iodine after, put under the 365nm ultraviolet light and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

Microscopical identification

Get this product content, put microscopically and observe: starch grain mostly is simple grain, class sphere or triangle, often assemble agglomerating, the omphalion herringbone, laminated striation is not obvious; How broken conduit is, and main is screw thread and scalariform duct, and vessel element is grown up; Prothenchyma (of wood), surface are seen and are class polygon or rectangle like, wall thickness, and pit is not obvious; Nonglandular hair, unicellular separate, faint yellow complete person is longer more, and tip is point, and is slightly crooked; The many bunchys of fiber, yellow or yellowish-brown, the libriform fiber microbend, terminal point or blunt circle have tangible single oblique pit; Stone cell, yellow, single loose from, be similar round, class is square, or it is dendritic to make irregular branch, similar round person wall is extremely thick, laminated striation is fine and closely woven, Kong Gouduo is not obvious; Divide the wall of dendritic stone cell thinner, cell is bigger; Pigment piece yellow or yellowish-brown are irregular shape more, are dispersed in; Oil cell is dispersed in, and is colourless or faint yellow, and the surface sight is similar round or class is square.

The experiment of limiting the quantity of of Aristolochic Acid in the raw material Asarum medicinal materials of SHANGTONGNING JIAONANG, concrete test method and result are as follows:

1, instrument and reagent

Instrument: Tianjin, island LC-10ATVP high performance liquid chromatograph; C18 (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. for Hypersil ODS200*4.6mm, 5 μ) chromatographic column.Reagent: methanol is chromatorgaphy reagent, and water is redistilled water, and other reagent are analytical pure.

Reference substance: Aristolochic Acid provides for Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and for assay usefulness, lot number is 0746-200103.

2, chromatographic condition

Mobile phase: the mobile phase of Aristolochic Acid assay method in the reference literature the results showed that with methanol-4% glacial acetic acid solution (55: 45) be that mobile phase is better.

Detect wavelength: through the reference substance solution of Aristolochic Acid is carried out UV scanning, its maximum absorption wavelength is at 315nm, so select 315nm as detecting wavelength.The UV scanning figure of Aristolochic Acid reference substance sees Fig. 1.

Flow velocity: 1.0ml/min.

Reference substance, test sample, Asarum medicinal materials chromatogram are seen Fig. 2,3,4 respectively.

3, the preparation of need testing solution

The extracting method of Aristolochic Acid assay in the reference literature, determine that the preparation method of this product need testing solution is as follows:

Get this product content 4.0g, accurate claim surely, put in the 100ml tool plug conical flask, add methanol 100ml, close plug claims decide weight, and supersound process 40 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methanol, shakes up filtration.The accurate subsequent filtrate 50.0ml that draws puts in the evaporating dish, and evaporate to dryness in the water-bath, residue add 0.1mol/L sodium hydroxide solution 30ml gradation dissolving, and be transferred in the separatory funnel, use chloroform extraction 6 times, each 20ml, discard chloroform liquid, aqueous alkali adds the 5mol/L hydrochloric acid solution and regulates about pH to 2 reuse chloroform extraction 4 times, each 20ml merges chloroform liquid, evaporate to dryness in the water-bath, residue adds the methanol gradation makes dissolving, puts in the 10ml measuring bottle, adds methanol to scale, shake up, filter, promptly.

4, the preparation of reference substance solution

Precision takes by weighing Aristolochic Acid reference substance 6mg, puts in the 50ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up, and precision is measured 5.0ml, puts in the 50ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets (containing Aristolochic Acid 12ug among every 1ml)

5, algoscopy

Accurate respectively reference substance solution, each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly.

6, linear relationship is investigated

Accurate absorption concentration is Aristolochic Acid reference substance solution 1,2,4,6,8, the 10 μ l sample introductions of 12 μ g/ml, measure its peak area integrated value, with sample size (ng) is abscissa, the peak area integrated value is a vertical coordinate, the drawing standard curve, its regression equation is: Y=2985.5427X+2943.633, r=0.99997.The result shows that Aristolochic Acid has good linear relationship in the 12-120ng scope.Data see Table 1.Canonical plotting is seen Fig. 5.

Table 1 linear relationship is investigated table as a result

7, lowest detectable limit

Accurate absorption concentration is the Aristolochic Acid reference substance solution 1.0ml of 12 μ g/ml, puts in the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up, and draws 10 μ l and injects chromatograph of liquid, measures, and the results are shown in Figure 6.The signal to noise ratio at Aristolochic Acid peak (S/N) is about 10, and lowest detection is limited to 1.2ng.

8, application of sample test

Get this product (lot number: 20040315) content 4.0g, accurate claim fixed, accurate respectively Aristolochic Acid reference substance solution (the 120 μ g/ml) 1.0ml that adds, i.e. 120 μ g, methanol 99ml measures Aristolochic Acid by above-mentioned inspection method, the results are shown in Figure 7.

The result shows that the Aristolochic Acid peak can separate preferably with other chromatographic peak in the sample.

9, finished product and medical material measurement result

9.1 medical material measurement result

Get Manchurian Wildginger 4.0g, the accurate title, decide, and proportionately the inspection method of Aristolochic Acid in the product quality standard is measured Aristolochic Acid, does not detect Aristolochic Acid in this product institute Herba Asari as a result, sees Fig. 4.

The source of the used Herba Asari of this product be the plant Herba Asari Asarum heterotropoides Fr.Schmidtvar.mandshuricum of Aristolochic Acid section (Maxim.) Kitan., dry herb.The place of production is Liaoning.

9.2 finished product measurement result

By the Aristolochic Acid in 6 batches of finished products of inspection method mensuration of drafting, the result does not all detect Aristolochic Acid.According to lowest detectable limit, the cold content of the Aristolochic Acid in this product is below 0.6ppm.

After comparing with reference substance solution, sample Aristolochic Acid peak shape and retention time and reference substance solution are close, and negative sample is noiseless.After " linear relationship experiment ", " lowest detectable limit ", " application of sample test ", prove that this method specificity is strong, exactly the content of Aristolochic Acid in the working sample.

The SHANGTONGNING JIAONANG that meets quality standard of the present invention has following drug effect:

SHANGTONGNING JIAONANG is the prescription of warming the meridian and promoting blood circulation, blood circulation promoting and blood stasis dispelling.Be used for the treatment of diseases such as traumatic injury, gastric abscess, dysmenorrhea more.Use the basic indication of this medicine to be: the injury is livid purple to be swollen and ache, dare not touch, or coldness and pain in the epigastrium, likes warm tenderness, light red tongue or purple, and the pulse condition string is puckery.These indications have embodied qi depression to blood stasis, and the pathogenesis of cold-evil's arthromyodynia passages through which vital energy circulates is so use very wide.As:

1, lumbar sprain and QI divergeny: show as injury pain, limitation of activity, aching as needle stabbing, or cyanosis of the skin are exerted and are hindered cough, breathe pain very, red tongue or dark, stringy pulse or puckery.The western medicine diagnosis exert wound, sprain, soft tissue injury, see above-mentioned shower, belong to this scope.

2, arthromyodynia: show as arthralgia as thorn, meet and tremble with fear more so, limitation of activity, happiness Wen Xinuan, very then swelling, light red tongue thready pulse.The rheumatic arthritis of western medicine diagnosis, traumatic arthritis see that above clinical manifestation person belongs to it.

3, gastric abscess: show as gastric abscess, warm tenderness is liked in gesture such as acupuncture, and stool is black in pitch, light red tongue or dark, and pulse condition stringy and thready pulse or string are puckery.The gastric ulcer of western medicine, duodenal bulbar ulcer etc. are seen above-mentioned shower, distinguish by this and control.

4, dysmenorrhea: show as menalgia, its color is purple black, passes the appointed time and causes, and stomachache as thorn are decided the place not move, and like warm tenderness, and tongue secretly has ecchymosis, and the pulse condition string is puckery.The menoxenia of western medicine diagnosis, adnexitis etc. are seen above-mentioned shower, all available it.

The invention has the advantages that can be by to the discriminating and the assay of plurality of raw materials medicine in the prescription, guarantees the drug effect and the stability of the SHANGTONGNING JIAONANG produced effectively, thereby can protect the safety of extensive patients medication better.

Specific embodiment

The discriminating of specific embodiment one Rhizoma Corydalis

Get this product content 3g, the 20ml that adds diethyl ether flooded 1 hour, and the ether solution that inclines adds strong ammonia solution 3ml and makes moisteningly, and the 20ml that adds diethyl ether dipping 3 hours filters, and filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, as need testing solution.Other gets the tetrahydropalmatine reference substance, adds dehydrated alcohol and makes the solution that contains 0.1mg among every 1ml, in contrast product solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene-acetone (17: 3) is developing solvent, launches, and takes out, dry, it is smoked clear to the speckle colour developing to put in the iodine steam, fling to iodine after, put under the ultraviolet light and inspect under (365nm).In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

Check three batch samples all up to specification, negative solution is noiseless.

Specific embodiment two microscopical identification

Get this product content, put microscopically and observe: starch grain mostly is simple grain, class sphere or triangle, often assemble agglomerating, the omphalion herringbone, laminated striation is not obvious; How broken conduit is, and main is screw thread and scalariform duct, and vessel element is grown up; Prothenchyma (of wood), surface are seen and are class polygon or rectangle like, wall thickness, and pit is not obvious; Nonglandular hair, unicellular separate, faint yellow complete person is longer more, and tip is point, and is slightly crooked; The many bunchys of fiber, yellow or yellowish-brown, the libriform fiber microbend, terminal point or blunt circle have tangible single oblique pit; Stone cell, yellow, single loose from, be similar round, class is square, or it is dendritic to make irregular branch, similar round person wall is extremely thick, laminated striation is fine and closely woven, Kong Gouduo is not obvious; Divide the wall of dendritic stone cell thinner, cell is bigger; Pigment piece yellow or yellowish-brown are irregular shape more, are dispersed in; Oil cell is dispersed in, and is colourless or faint yellow, and the surface sight is similar round or class is square.

The assay of Aristolochic Acid in specific embodiment three Herba Asaris

Assay is according to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler, is mobile phase with methanol-4% glacial acetic acid solution (55: 45); The detection wavelength is 315nm, and number of theoretical plate should be not less than 3000 by the Aristolochic Acid peak;

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Aristolochic Acid reference substance, adds methanol and make the solution that contains 10ug among every 1ml, promptly;

The preparation of need testing solution: get this product content 4.0g, accurate claim surely, put in the 100ml tool plug conical flask, add methanol 100ml, close plug claims decide weight, and supersound process 40 minutes is put coldly, claims to decide weight again, supplies with methanol to subtract the important of mistake, shakes up filtration; The accurate subsequent filtrate 50.0ml that draws puts in the evaporating dish, and evaporate to dryness in the water-bath, residue add 0.1mol/L sodium hydroxide solution 30ml gradation dissolving, and be transferred in the separatory funnel, use chloroform extraction 6 times, each 20ml, discard chloroform liquid, aqueous alkali adds the 5mol/L hydrochloric acid solution and regulates about PH to 2 reuse chloroform extraction 4 times, each 20ml merges chloroform, evaporate to dryness in the water-bath, residue adds the methanol gradation makes dissolving, puts in the 10ml measuring bottle, adds methanol to scale, shake up, filter, promptly;

Algoscopy: accurate respectively reference substance solution and each 10ul of need testing solution of drawing, inject chromatograph of liquid, measure.

Use said method, must not detect Aristolochic Acid.

Description of drawings:

1, (1) Fig. 1 is Aristolochic Acid reference substance UV scanning figure;

(2) Fig. 2 is an Aristolochic Acid reference substance HPLC chromatogram;

(3) Fig. 3 is a SHANGTONGNING JIAONANG test sample HPLC chromatogram;

(4) Fig. 4 is an Asarum medicinal materials HPLC chromatogram;

(5) Fig. 5 is an Aristolochic Acid reference substance canonical plotting;

(6) Fig. 6 is an Aristolochic Acid reference substance lowest detectable limit HPLC chromatogram;

(7) Fig. 7 adds the HPLC chromatogram of Aristolochic Acid reference substance for the SHANGTONGNING JIAONANG test sample.

Claims (7)

1, a kind of method of quality control for the treatment of grieved Chinese medicine preparation, this Chinese medicine preparation is prepared by crude drug Olibanum (processed), Myrrha (processed), Radix Et Rhizoma Nardostachyos, Rhizoma Corydalis (processed with vinegar), Herba Asari, Rhizoma Cyperi (processed), Rhizoma Kaempferiae, the Radix Angelicae Dahuricae, it is characterized in that carrying out quality control by the determination limit to Aristolochic Acid in the discriminating of Rhizoma Corydalis and/or microscopical identification and/or the Herba Asari.

2, a kind of method of quality control for the treatment of grieved Chinese medicine preparation as claimed in claim 1 is characterized in that described Chinese medicine preparation is a SHANGTONGNING JIAONANG.

3, a kind of method of quality control for the treatment of grieved Chinese medicine preparation as claimed in claim 1, the thin layer chromatography that it is characterized in that its used Rhizoma Corydalis are to realize controlling the purpose of drug quality by implementing following concrete discrimination method:

The discriminating of Rhizoma Corydalis

Get this product content 1.5-4.5g, the 10-30ml that adds diethyl ether flooded 0.5-1.5 hour, and ether solution inclines, add strong ammonia solution 1.5-4.5ml and make moisteningly, the 10-30ml that adds diethyl ether dipping 1.5-4.5 hour filters, filtrate evaporate to dryness, residue add dehydrated alcohol 0.5-1.5ml makes dissolving, as need testing solution; Other gets the tetrahydropalmatine reference substance, adds dehydrated alcohol and makes the solution that contains 0.05-0.15mg among every 0.5-1.5ml, in contrast product solution; Test according to thin layer chromatography, draw each 2.5-7.5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, benzene-the acetone that with the ratio is 8-25: 1.5-4.5 is developing solvent, launches, and takes out, dry, it is smoked clear to the speckle colour developing to put in the iodine steam, fling to iodine after, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

4, a kind of method of quality control for the treatment of grieved Chinese medicine preparation as claimed in claim 3, the thin layer chromatography that it is characterized in that its used Rhizoma Corydalis are to realize controlling the purpose of drug quality by implementing following concrete discrimination method:

The discriminating of Rhizoma Corydalis

Get this product content 3g, the 20ml that adds diethyl ether flooded 1 hour, and the ether solution that inclines adds strong ammonia solution 3ml and makes moisteningly, and the 20ml that adds diethyl ether dipping 3 hours filters, and filtrate evaporate to dryness, residue add dehydrated alcohol 1ml makes dissolving, as need testing solution; Other gets the tetrahydropalmatine reference substance, adds dehydrated alcohol and makes the solution that contains 0.1mg among every 1ml, in contrast product solution; According to thin layer chromatography test, draw each 5 μ 1 of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be that benzene-acetone of 17: 3 is developing solvent with ratio, launch, take out, dry, it is smoked clear to the speckle colour developing to put in the iodine steam, fling to iodine after, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.

5, a kind of method of quality control for the treatment of grieved Chinese medicine preparation as claimed in claim 1 is characterized in that its used microscopical identification is by implementing the purpose that following method realizes controlling drug quality:

Get this product content, put microscopically and observe: starch grain mostly is simple grain, class sphere or triangle, often assemble agglomerating, the omphalion herringbone, laminated striation is not obvious; How broken conduit is, and main is screw thread and scalariform duct, and vessel element is grown up; Prothenchyma (of wood), surface are seen and are class polygon or rectangle like, wall thickness, and pit is not obvious; Nonglandular hair, unicellular separate, faint yellow complete person is longer more, and tip is point, and is slightly crooked; The many bunchys of fiber, yellow or yellowish-brown, the libriform fiber microbend, terminal point or blunt circle have tangible single oblique pit; Stone cell, yellow, single loose from, be similar round, class is square, or it is dendritic to make irregular branch, similar round person wall is extremely thick, laminated striation is fine and closely woven, Kong Gouduo is not obvious; Divide the wall of dendritic stone cell thinner, cell is bigger; Pigment piece yellow or yellowish-brown are irregular shape more, are dispersed in; Oil cell is dispersed in, and is colourless or faint yellow, and the surface sight is similar round or class is square.

6,, it is characterized in that its determination limit to Aristolochic Acid in the Herba Asari adopts following method to realize as the described a kind of method of quality control for the treatment of grieved Chinese medicine preparation of claim 1-5:

Assay is according to high effective liquid chromatography for measuring;

The test of chromatographic condition and system suitability: with octadecylsilane chemically bonded silica is filler, is that 55: 45 methanol-4% glacial acetic acid solution is a mobile phase with ratio; The detection wavelength is 315nm, and number of theoretical plate should be not less than 3000 by the Aristolochic Acid peak;

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Aristolochic Acid reference substance, adds methanol and make the solution that contains 10ug among every 1ml, promptly;

The preparation of need testing solution: get this product content 2.0-6.0g, the accurate title, decide, and puts in the 100ml tool plug conical flask, adds methanol 50-150ml, close plug claims to decide weight, supersound process 20-60 minute, puts cold, weight decided in title again, supplies with methanol to subtract the important of mistake, shakes up, and filters; The accurate subsequent filtrate 25-75ml that draws, put in the evaporating dish, evaporate to dryness in the water-bath, residue add 0.05-0.15mol/L sodium hydroxide solution 15-45ml gradation dissolving, and are transferred in the separatory funnel, with chloroform extraction 3-9 time, each 10-30ml discards chloroform liquid, and aqueous alkali adds the 2.5-7.5mol/L hydrochloric acid solution to be regulated about PH to 1-3, reuse chloroform extraction 2-6 time, each 10-30ml merges chloroform, evaporate to dryness in the water-bath, residue adds the methanol gradation makes dissolving, put in the 10ml measuring bottle, add methanol, shake up to scale, filter, promptly;

Algoscopy: accurate respectively reference substance solution and each 5-15ul of need testing solution of drawing, inject chromatograph of liquid, measure.

7, a kind of method of quality control for the treatment of grieved Chinese medicine preparation as claimed in claim 6 is characterized in that the following method of its employing limits the content of Aristolochic Acid in the Herba Asari:

Assay is according to high effective liquid chromatography for measuring;

The test of chromatographic condition and system suitability: with octadecylsilane chemically bonded silica is filler, is that 55: 45 methanol-4% glacial acetic acid solution is a mobile phase with ratio; The detection wavelength is 315nm, and number of theoretical plate should be not less than 3000 by the Aristolochic Acid peak;

The preparation of reference substance solution: it is an amount of that precision takes by weighing the Aristolochic Acid reference substance, adds methanol and make the solution that contains 10ug among every 1ml, promptly;

The preparation of need testing solution: get this product content 4.0g, accurate claim surely, put in the 100ml tool plug conical flask, add methanol 100ml, close plug claims decide weight, and supersound process 40 minutes is put coldly, claims to decide weight again, supplies with methanol to subtract the important of mistake, shakes up filtration; The accurate subsequent filtrate 50.0ml that draws puts in the evaporating dish, and evaporate to dryness in the water-bath, residue add 0.1mol/L sodium hydroxide solution 30ml gradation dissolving, and be transferred in the separatory funnel, use chloroform extraction 6 times, each 20ml, discard chloroform liquid, aqueous alkali adds the 5mol/L hydrochloric acid solution and regulates about PH to 2 reuse chloroform extraction 4 times, each 20ml merges chloroform, evaporate to dryness in the water-bath, residue adds the methanol gradation makes dissolving, puts in the 10ml measuring bottle, adds methanol to scale, shake up, filter, promptly;

Algoscopy: accurate respectively reference substance solution and each 10ul of need testing solution of drawing, inject chromatograph of liquid, measure;

Use said method, must not detect Aristolochic Acid.

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