CN101036774B

CN101036774B - Quality control method of compound cantharidin oral preparations

Info

Publication number: CN101036774B
Application number: CN2007102004651A
Authority: CN
Inventors: 叶湘武; 汤琼; 李磊
Original assignee: Guizhou Yibai Pharmaceutical Co Ltd
Current assignee: Guizhou Yibai Pharmaceutical Co Ltd
Priority date: 2007-04-17
Filing date: 2007-04-17
Publication date: 2010-12-29
Anticipated expiration: 2027-04-17
Also published as: CN101036774A

Abstract

The invention relates to a quality control method for compound mylabris oral preparation, comprising character, identification, inspection and partly or whole items of assay, wherein the identification comprising TLC identification of pannax, astragalus, barbat skullcap, cornel and glossy privet fruit; the items of inspection comprising water content, the residual quantity of chloroform and the other routine items; the assay being of astragalus and cantharidim in mylabris. Compared with existing technology, the quality control method complementes the assay methods of astragalus and cantharidim, the TLC identification of barbat skullcap, cornel and glossy privet fruit and the inspection of the residual quantity of chloroform. The quality control method also improves the TLC identification of pannax and astragalus, and enhances the quality control standard of compound mylabris oral preparation, and effectively control the product quality, therefore the clinical effect is insured.

Description

A kind of detection method of compound cantharidin oral preparations

Technical field: the present invention relates to a kind of method of quality control of compound cantharidin oral preparations, belong to the technical field of medicine being carried out quality control.

Background technology: malignant tumour is a kind of disease of serious harm human health, and is human in decades always in continuous growth of researching and developing new medicine with effective inhibition malignant tumour.In research process, effectively filter out medicine and just seem particularly important with antitumor action.Compound cantharidin oral preparations by Chinese blister beetle, genseng, the Radix Astragali, wilsonii, triangular, Sculellaria barbata, curcuma zedoary, the fruit of medicinal cornel, the fruit of glossy privet, bear gall powder, Radix Glycyrrhizae totally ten simply medication preparation form, has the broken blood stasis of blood that disappears, attack the effect of malicious phagedenoma, be mainly used in diseases such as primary carcinoma of liver, lung cancer, the carcinoma of the rectum, malignant lymphoma, gynecologic malignant tumor.Wherein " cantharides capsule " is on the books in the 17 in health ministry drug standards Chinese traditional patent formulation preparation.But through discovering, shortcoming such as it is simple that existing compound mylabris preparation exists quality control standard, and product quality is wayward.Sculellaria barbata, the fruit of medicinal cornel, the fruit of glossy privet etc. are effective constituent in the compound mylabris preparation, and existing preparation quality standard is not with its thin layer discrimination method income wherein; During the thin layer of genseng and the Radix Astragali two flavor medicinal materials differentiated, the selection of developping agent was not ideal enough, causes standing time longer, and operation inconvenience, and because factor affecting such as operating environments causes in discrimination process degree of separation bad, and the spot colour developing is clear inadequately; Chloroform toxicity is bigger, is two kind solvents that country lists, and existing preparation extraction process is to carry out lixiviate with chloroform, and is not strict with its limit of control in quality standard.In addition, existing preparation quality standard is not set up assay method accurately and effectively to the content of the effective constituent Radix Astragali and cantharidin in the compound mylabris preparation.So existing method of quality control can not effectively be controlled the quality of compound cantharidin oral preparations, thereby will influence the clinical efficacy of said preparation.But how formulating scientific and reasonable method of quality control, effectively control the quality of this oral formulations, thereby guarantee its clinical efficacy, is the problem that those skilled in the art must consider.

Summary of the invention:

The objective of the invention is to: a kind of method of quality control of compound cantharidin oral preparations is provided, and this oral formulations comprises capsule, tablet, granule.The present invention is directed to the deficiencies in the prior art, method of quality control to compound cantharidin oral preparations is studied, the thin layer that has increased the assay of the Radix Astragali and cantharidin and Sculellaria barbata, the fruit of medicinal cornel, the fruit of glossy privet is differentiated project, increased the inspection of chloroform residual quantity, and the thin layer discrimination method of genseng and the Radix Astragali two flavor medicinal materials improved, thickness to developping agent and thin layer plate is selected, optimized discrimination method, improve the quality control standard of this compound cantharidin oral preparations, thereby guaranteed the clinical efficacy of said preparation.

Compound cantharidin oral preparations of the present invention is to constitute like this: calculate according to composition by weight: it mainly is prepared from by Chinese blister beetle 4 12, genseng 10-30, Radix Astragali 50-150, wilsonii 50-150, triangular 16-48, Sculellaria barbata 60-180, curcuma zedoary 16-48, fruit of medicinal cornel 20-60, fruit of glossy privet 20-60, bear gall powder 0.4-1.2 and Radix Glycyrrhizae 10-30.Its preparation method is: above ten simply, and except that bear gall powder, genseng, the fruit of medicinal cornel, the fruit of glossy privet, Sculellaria barbata are ground into fine powder, sieve, and be standby; Chinese blister beetle is soaked with chloroform and extracts 1-5 time, and each 16-48ml soaked 48-96 hour, merged extract, reclaimed chloroform, was concentrated into the thick paste shape; Five tastes boilings such as all the other Radixs Astragali 1-5 time, each 0.5-5 hour, collecting decoction filtered, and filtrate is concentrated into relative density and is about 1.00-1.400 (60-100 ℃); Bear gall powder adds fine powders such as above-mentioned thick paste, concentrate and genseng after adding 80 ℃ of water-soluble separating, and stirs evenly, and in oven dry below 100 ℃, is ground into fine powder, adds auxiliary material, makes capsule, tablet or granule with conventional method.Auxiliary material can be added in this preparation, also auxiliary material can be do not added.

Method of quality control of the present invention mainly comprise in proterties, discriminating, inspection and the assay project partly or entirely; Wherein differentiate the thin-layer chromatography discriminating that comprises genseng, the Radix Astragali, Sculellaria barbata, the fruit of medicinal cornel and the fruit of glossy privet in the preparation; Inspection comprises moisture, chloroform residual quantity and other conventional projects; Assay is the assay to contained cantharidin in the Radix Astragali in the preparation and the Chinese blister beetle.

The discrimination method of the genseng and the Radix Astragali is to be contrast with ginsenoside Rg1's reference substance and Astragaloside IV reference substance, and with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is the thin-layered chromatography of developping agent; The discrimination method of Sculellaria barbata is to be contrast with the Sculellaria barbata control medicinal material, and with toluene: ethyl formate: formic acid=2-20: 1-9: 1-9 is the thin-layered chromatography of developping agent; The discrimination method of the fruit of medicinal cornel is to be contrast with the loganin reference substance, and with toluene: acetone: absolute ethyl alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is the thin-layered chromatography of developping agent; The discrimination method of the fruit of glossy privet is to be contrast with fruit of glossy privet control medicinal material, and with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is the thin-layered chromatography of developping agent; The inspection method of chloroform residual quantity is to be the vapor-phase chromatography of contrast with the chloroform reference substance; The content assaying method of the Radix Astragali is to be contrast with the Astragaloside IV reference substance, is the high performance liquid chromatography of moving phase with acetonitrile: water=10-90: 90-10; The content assaying method of contained cantharidin is to be the vapor-phase chromatography of contrast with the cantharidin reference substance in the Chinese blister beetle.

Described discrimination method comprises the part or all of of following project:

(1) gets capsule, tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put refluxing extraction in the water-bath, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put refluxing extraction in the water-bath, reclaim methyl alcohol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separating funnel, wash container again with water, be transferred in the separating funnel, extract with water saturated normal butyl alcohol, extract washes with water, divide and get normal butyl alcohol liquid, evaporate to dryness, the dissolving of residue water is transferred on the neutral alumina post, use earlier the chloroform wash-out, discard the chloroform eluent, use methanol-eluted fractions again, eluent is evaporate to dryness in water-bath, residue adds methyl alcohol makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is developping agent (controlled humidity in case of necessity), launches, and takes 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 ℃, puts under the 200-500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(2) get capsule, tablet or granule, porphyrize, the reflux that adds diethyl ether is put coldly, filters, and discards filtrate, and the dregs of a decoction add the methyl alcohol reflux, put coldly, filter, the filtrate evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Other gets the sun plant control medicinal material, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution and control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl formate: formic acid=2-20: 1-9: 1-9 is a developping agent, launch, take out, dry; Put under the 200-500nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;

(3) get capsule, tablet or granule, porphyrize adds methyl alcohol, and reflux is put cold, filter, filtrate evaporate to dryness, residue add methyl alcohol and dissolve in right amount, are added on the neutral alumina post, use methanol-eluted fractions, collect eluent, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the loganin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is a developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;

(4) get capsule, tablet or granule, porphyrize adds ethyl acetate, and is ultrasonic, filter, and the filtrate evaporate to dryness, residue adds anhydrous alcohol solution, as need testing solution; Other gets fruit of glossy privet control medicinal material, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution and control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is a 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

Discrimination method comprises the part or all of of following project more specifically:

(1) gets capsule, each 2-5g of tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put in the water-bath refluxing extraction 1-3 hour, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put in the water-bath refluxing extraction 2-6 hour, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 1-10ml makes dissolving, goes in the separating funnel, uses 1-3 washing container of 1-10ml moisture again, be transferred in the separating funnel, extract 2-6 time with water saturated normal butyl alcohol, each 2-20ml merges normal butyl alcohol liquid, wash with water 1-3 time, each 1-10ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 1-5 time with 0.4-1.2ml moisture, be transferred to 2g, dry column-packing, diameter 1cm, long 25cm, on 100～200 purpose neutral alumina posts, with 10-50ml chloroform wash-out, discard the chloroform eluent earlier, use 70% methyl alcohol 10-40ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 0.5-2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 5-20 μ l, reference substance solution 2-10 μ l puts respectively on the thick same silica gel g thin-layer plate of 450-550 μ m, and with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is developping agent (controlled humidity in case of necessity), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 200-500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(2) get capsule, tablet or granule 1-4g, porphyrize, the 10-40ml that adds diethyl ether reflux 10-50 minute, is put cold, filter, discard filtrate, the dregs of a decoction add methyl alcohol 10-40ml, reflux 10-50 minute, put cold, filter, filtrate evaporate to dryness, residue add methyl alcohol 0.5-2ml dissolving, as need testing solution; Other gets sun plant control medicinal material 0.5-2g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 2-20 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl formate: formic acid=2-20: 1-9: 1-9 is a developping agent, launch, take out, dry; Put under the 200-500nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;

(3) get capsule, tablet or granule 1-5g, porphyrize adds 50% methyl alcohol 10-50ml, reflux 0.5-2 hour, put coldly, filter, the filtrate evaporate to dryness, residue adds 50% methyl alcohol and dissolves in right amount, is added on 4g, 100～200 purpose neutral alumina posts, with 40% methyl alcohol 20-80ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 0.5-2ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 5-20 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is a developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;

(4) get capsule, tablet or granule 1-5g, porphyrize adds ethyl acetate 10-40ml, and ultrasonic 10-30min filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol 0.5-2ml dissolving, as need testing solution; Other gets fruit of glossy privet control medicinal material 0.2-2g, shines medicinal material solution in pairs with legal system; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 2-20 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is a 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

The inspection method of chloroform residual quantity is: shine gas chromatography determination: the fused-silica capillary column Rtx-5 of column length 30m, internal diameter 0.25mm, thickness 0.25 μ m is a stationary phase with 5% phenyl methyl siloxane; Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100-120 ℃, heating 25-40min, 110-120 ℃ of sample introduction needle temperature, 110-130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and dinethylformamide (DMF) dissolving promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide (DMF), close plug, jolting, centrifugal, get supernatant, promptly get need testing solution; Accurate respectively absorption reference substance solution and need testing solution roll lid in the head space bottle, after high temperature balance a period of time, get saturated gas inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%.

The inspection method of chloroform residual quantity is more specifically: shine gas chromatography determination: the fused-silica capillary column Rtx-5 of column length 30m, internal diameter 0.25mm, thickness 0.25 μ m is a stationary phase with 5% phenyl methyl siloxane; Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100-120 ℃, heating 25-40min, 110-120 ℃ of sample introduction needle temperature, 110-130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the solution that every 1mL contains 10-30 μ g is made in dinethylformamide (DMF) dissolving, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, get the about 0.5-2.0g of content, the accurate title, decide, and puts in the tool plug test tube, accurate 75%N, N dimethyl formamide (DMF) 5-20ml, close plug, the jolting 1-5min of adding, centrifugal, get supernatant, promptly get need testing solution; Respectively accurate reference substance solution and each 1-4ml of need testing solution of drawing rolls lid in 20ml head space bottle, behind 110 ℃ of balance 20-50min, get saturated gas 0.5-2.0ml inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%.

Described content assaying method comprises the part or all of of following project:

(1) Radix Astragali shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with acetonitrile: water=10-90: 90-10; Flow velocity 0.5-1.0ml/min; Column temperature 30-60 ℃; The evaporative light-scattering detector drift tube temperature is 80-100 ℃; Atomization gas is an air, and flow velocity is 1.5-3.0L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds dissolve with methanol, shakes up, and promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, accurate claim surely, put in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution, reflux, extract evaporate to dryness, residue add and are transferred in the separating funnel after water makes dissolving, use ethyl acetate extraction; Add water washing, discard acetic acid ethyl fluid, merge water liquid, extract with water saturated normal butyl alcohol; Extract the back with the ammonia solution washing, discard ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue adds water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins water wash-out, discard water liquid, use 70% ethanol elution again, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in the measuring bottle, add methyl alcohol to scale, shake up, filter with miillpore filter, promptly get need testing solution; The accurate respectively reference substance solution 1-10 μ l of absorption and 2-20 μ l, need testing solution 2-20 μ l inject hplc determination content; In this oral formulations, contain the Radix Astragali in the capsule with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.01mg/g;

(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 immobile liquid is 1.5%, carrier Shimalite W (AW-DMCS) 80-100 order; Fid detector, injector temperature: 230-250 ℃; Detector temperature: 230-250 ℃; Column temperature is 150-160 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and adds the chloroform dissolving, shakes up, and promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, the accurate title, decide, and puts in the beaker, adds the sodium chloride sodium hydroxide solution, add acetone again, ultrasonic, put cold, add concentrated hydrochloric acid, stir, leave standstill, put coldly, suction filtration washs residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction, and chloroform solution is put 50 ℃ of water-bath Back stroke to a small amount of (chloroform must not be volatilized in the whole operation process), add the chloroform constant volume again, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 0.5-5 μ l of need testing solution respectively, and inject gas chromatograph is measured content; In this oral formulations, contain cantharidin C in the capsule ₁₀H ₁₂O ₄Be 0.025-0.180mg/g; Contain cantharidin C in the tablet ₁₀H ₁₂O ₄Be 0.025-0.180mg/g; Contain cantharidin C in the granule ₁₀H ₁₂O ₄Be 0.003-0.018mg/g.

Content assaying method comprises the part or all of of following project more specifically:

(1) Radix Astragali shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with acetonitrile: water=10-90: 90-10; Flow velocity 0.5-1.0ml/min; Column temperature 30-60 ℃; The evaporative light-scattering detector drift tube temperature is 80-100 ℃; Atomization gas is an air, and flow velocity is 1.5-3.0L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.05-0.5mg, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, porphyrize, get about 0.5-2g, the accurate title, decide, and puts in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution 50-100ml, reflux 4-6 hour, extract evaporate to dryness, residue added and are transferred in the separating funnel after water 10-40ml makes dissolving, with ethyl acetate extraction 1-3 time, each 10-40ml; Combined ethyl acetate liquid adds water 10-20ml washing; Discard acetic acid ethyl fluid, merge water liquid, extract 3-5 time, each 20-60ml with water saturated normal butyl alcohol; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 20-60ml washing 1-2 time, normal butyl alcohol liquid evaporate to dryness, residue add water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins of internal diameter 1.5cm, long 12cm, water 30-70ml wash-out discards water liquid, uses 70% ethanol 60-100ml wash-out again, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, filter, promptly get need testing solution with 0.45 μ m miillpore filter; The accurate respectively reference substance solution 1-10 μ l of absorption and 2-20 μ l, need testing solution 2-20 μ l inject hplc determination content; In this oral formulations, contain the Radix Astragali in the capsule with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.01mg/g;

(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 50% methyl-50% phenyl polysiloxane immobile liquid is 1.5%, carrier Shimalite W (AW-DMCS) 80-100 order; Fid detector, injector temperature: 230-250 ℃; Detector temperature: 230-250 ℃; Column temperature is 150-160 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 30-50 μ g, promptly gets reference substance solution; Get the capsule under the content uniformity item, tablet or granule content, porphyrize is got about 1-3g, the accurate title, decide, and puts in the 100ml beaker, adds sodium chloride sodium hydroxide solution 30-50ml, add 0.5-2ml acetone again, power 250w, under the frequency 20kHz ultrasonic 20-50 minute, put cold, add the 2-8ml concentrated hydrochloric acid, stir, leave standstill, put cold, suction filtration, wash residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction 2-4 time, each 30-40ml, combined chloroform liquid is put 50 ℃ of water-bath Back stroke to 1～2ml (chloroform must not be volatilized in the whole operation process), adds chloroform again and is settled to 3ml, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 0.5-5 μ l of need testing solution respectively, and inject gas chromatograph is measured content; In this oral formulations, contain cantharidin C in the capsule ₁₀H ₁₂O ₄Be 0.025-0.180mg/g; Contain cantharidin C in the tablet ₁₀H ₁₂O ₄Be 0.025-0.180mg/g; Contain cantharidin C in the granule ₁₀H ₁₂O ₄Be 0.003-0.018mg/g.

Method of quality control of the present invention comprises:

Proterties: for capsule, content is that yellow green is to tan powder, mildly bitter flavor Hui Tian;

For tablet, product is a Film coated tablets, removes to show yellow green behind the film-coating to sepia, mildly bitter flavor Hui Tian;

For granule, product is that pale brown look is to auburn particle; It is sweet to distinguish the flavor of;

Differentiate: (1) gets capsule, tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put refluxing extraction in the water-bath, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put refluxing extraction in the water-bath, reclaim methyl alcohol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separating funnel, wash container again with water, be transferred in the separating funnel, extract with water saturated normal butyl alcohol, extract washes with water, divide and get normal butyl alcohol liquid, evaporate to dryness, the dissolving of residue water is transferred on the neutral alumina post, use earlier the chloroform wash-out, discard the chloroform eluent, use methanol-eluted fractions again, eluent is evaporate to dryness in water-bath, residue adds methyl alcohol makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is developping agent (controlled humidity in case of necessity), launches, and takes 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 ℃, puts under the 200-500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(4) get capsule, tablet or granule, porphyrize adds ethyl acetate, and is ultrasonic, filter, and the filtrate evaporate to dryness, residue adds anhydrous alcohol solution, as need testing solution; Other gets fruit of glossy privet control medicinal material, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution and control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is a 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;

Check: moisture must not cross 15.0%;

The chloroform residual quantity is shone gas chromatography determination: the fused-silica capillary column Rtx-5 of column length 30m, internal diameter 0.25mm, thickness 0.25 μ m is a stationary phase with 5% phenyl methyl siloxane; Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100-120 ℃, heating 25-40min, 110-120 ℃ of sample introduction needle temperature, 110-130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and dinethylformamide (DMF) dissolving promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide (DMF), close plug, jolting, centrifugal, get supernatant, promptly get need testing solution; Accurate respectively absorption reference substance solution and need testing solution roll lid in the head space bottle, after high temperature balance a period of time, get saturated gas inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%;

Other capsules of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the relevant regulations under capsule, tablet or the granule item;

Assay: (1) Radix Astragali shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with acetonitrile: water=10-90: 90-10; Flow velocity 0.5-1.0ml/min; Column temperature 30-60 ℃; The evaporative light-scattering detector drift tube temperature is 80-100 ℃; Atomization gas is an air, and flow velocity is 1.5-3.0L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds dissolve with methanol, shakes up, and promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, accurate claim surely, put in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution, reflux, extract evaporate to dryness, residue add and are transferred in the separating funnel after water makes dissolving, use ethyl acetate extraction; Add water washing, discard acetic acid ethyl fluid, merge water liquid, extract with water saturated normal butyl alcohol; Extract the back with the ammonia solution washing, discard ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue adds water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins water wash-out, discard water liquid, use 70% ethanol elution again, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in the measuring bottle, add methyl alcohol to scale, shake up, filter with miillpore filter, promptly get need testing solution; The accurate respectively reference substance solution 1-10 μ l of absorption and 2-20 μ l, need testing solution 2-20 μ l inject hplc determination content; In this oral formulations, contain the Radix Astragali in the capsule with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.01mg/g;

The applicant finds after deliberation, adopts the quality of following method of quality control with easier control compound cantharidin oral preparations, is more conducive to guarantee the clinical efficacy of said preparation.So described method of quality control also can comprise:

Differentiate: (1) gets capsule, each 2 5g of tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put in the water-bath refluxing extraction 1-3 hour, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put in the water-bath refluxing extraction 2-6 hour, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 1-10ml makes dissolving, goes in the separating funnel, uses 1-3 washing container of 1-10ml moisture again, be transferred in the separating funnel, extract 2-6 time with water saturated normal butyl alcohol, each 2-20ml merges normal butyl alcohol liquid, wash with water 1-3 time, each 1-10ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 1-5 time with 0.4-1.2ml moisture, be transferred to 2g, dry column-packing, diameter 1cm, long 25cm, on 100～200 purpose neutral alumina posts, with 10-50ml chloroform wash-out, discard the chloroform eluent earlier, use 70% methyl alcohol 10-40ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 0.5-2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 5-20 μ l, reference substance solution 2-10 μ l puts respectively on the thick same silica gel g thin-layer plate of 450-550 μ m, and with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is developping agent (controlled humidity in case of necessity), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 200-500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(4) get capsule, tablet or granule 1-5g, porphyrize adds ethyl acetate 10-40ml, and ultrasonic 10-30min filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol 0.5-2ml dissolving, as need testing solution; Other gets fruit of glossy privet control medicinal material 0.2-2g, shines medicinal material solution in pairs with legal system; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 2-20 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is a 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;

Check: moisture must not cross 15.0%;

The chloroform residual quantity is shone gas chromatography determination: the fused-silica capillary column Rtx-5 of column length 30m, internal diameter 0.25mm, thickness 0.25 μ m is a stationary phase with 5% phenyl-methylsiloxane; Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100-120 ℃, heating 25-40min, 110-120 ℃ of sample introduction needle temperature, 110-130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the solution that every 1mL contains 10-30 μ g is made in dinethylformamide (DMF) dissolving, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, get the about 0.5-2.0g of content, the accurate title, decide, and puts in the tool plug test tube, accurate 75%N, dinethylformamide (DMF) 5-20ml, close plug, the jolting 1-5min of adding, centrifugal, get supernatant, promptly get need testing solution; Respectively accurate reference substance solution and each 1-4ml of need testing solution of drawing rolls lid in 20ml head space bottle, behind 110 ℃ of balance 20-50min, get saturated gas 0.5-2.0ml inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%;

Assay: (1) Radix Astragali shines high effective liquid chromatography for measuring: chromatographic column is C18 or C4 or C8 post, is moving phase with acetonitrile: water=10-90: 90-10; Flow velocity 0.5-1.0ml/min; Column temperature 30-60 ℃; The evaporative light-scattering detector drift tube temperature is 80-100 ℃; Atomization gas is an air, and flow velocity is 1.5-3.0L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.05-0.5mg, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, porphyrize, get about 0.5-2g, the accurate title, decide, and puts in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution 50-100ml, reflux 4-6 hour, extract evaporate to dryness, residue added and are transferred in the separating funnel after water 10-40ml makes dissolving, with ethyl acetate extraction 1-3 time, each 10-40ml; Combined ethyl acetate liquid adds water 10-20ml washing; Discard acetic acid ethyl fluid, merge water liquid, extract 3-5 time, each 20-60ml with water saturated normal butyl alcohol; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 20-60ml washing 1-2 time, normal butyl alcohol liquid evaporate to dryness, residue add water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins of internal diameter 1.5cm, long 12cm, water 30-70ml wash-out discards water liquid, uses 70% ethanol 60-100ml wash-out again, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, filter, promptly get need testing solution with 0.45 μ m miillpore filter; The accurate respectively reference substance solution 1-10 μ l of absorption and 2-20 μ l, need testing solution 2-20 μ l inject hplc determination content; In this oral formulations, contain the Radix Astragali in the capsule with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with Astragaloside IV C ₄₁H ₆₈O ₁₄Meter must not be less than 0.01mg/g;

More than used sodium chloride sodium hydroxide solution be in 1mol/L sodium hydroxide solution 100mL, to add sodium chloride 2g, stir, dissolving forms.

Method of quality control of the present invention is the preferred plan that obtains through a large amount of shaker tests, and following experimental study is a preferred process of the present invention:

One, the research of Radix Astragali content assaying method

Relatively poor because of the Astragaloside IV uv absorption, belong to end and absorb, if detect with UV-detector, then its sensitivity is low, disturbs greatly, reappearance is relatively poor, especially for compound preparation, measures its content difficulty more.In recent years, the bibliographical information that uses HPLC ELSD method to measure Astragaloside content is arranged, its sensitivity, stability and reappearance all can meet the requirement of assay.This test is with reference to the experiment condition in the documents and materials, and in conjunction with " Chinese pharmacopoeia has been done suitable adjustment about the content assaying method of Astragaloside IV in the Milkvetch Root to chromatographic condition, and theoretical cam curve (N) press the calculating of Astragaloside IV peak more than 3000.

(1) need testing solution preparation method research:

1, determining of extracting method:

Method 1: the product of getting it filled, porphyrize is put in the apparatus,Soxhlet's, add 1% NaOH ethanolic solution, extract the water bath method extract in the water-bath heating, add water, low-grade fever makes dissolving, puts the extraction that adds methylene chloride in the separating funnel, abandon dichloromethane solution, the aqueous solution extracted by ether is abandoned ether solution, aqueous solution is extracted with water saturated normal butyl alcohol, butanol solution washes with water, abandons water layer, washs with 1% potassium dihydrogen phosphate again, abandon water layer, n-butanol extracting liquid is put water bath method, and residue adds dissolve with methanol, and quantitatively is transferred in the measuring bottle, be diluted to scale with methyl alcohol, shake up, filter with miillpore filter (0.45 μ m), promptly.

Method 2: the product of getting it filled, porphyrize is got fine powder and is put in the apparatus,Soxhlet's, adds the methyl alcohol heating and refluxing extraction, the methanol solution evaporate to dryness, residue adds water, and low-grade fever makes dissolving, water liquid extracts with water-saturated n-butanol, and normal butyl alcohol liquid washs with 2% potassium hydroxide solution, discards alkali lye, with the washing of normal butyl alcohol saturation water, discard water liquid, the n-butanol layer evaporate to dryness again, residue adds methyl alcohol makes dissolving, and quantitatively is transferred in the measuring bottle, is diluted to scale with methyl alcohol, shake up, filter with miillpore filter (0.45 μ m), promptly.

Method 3: the product of getting it filled, porphyrize is got fine powder and added methyl alcohol, and is ultrasonic, pour out supernatant liquid filtering, it is ultrasonic that residue adds methyl alcohol, filters, and merges filtered fluid, evaporate to dryness, residue is dissolved in water, and extracts with methylene chloride, water liquid extracts with water-saturated n-butanol, and normal butyl alcohol liquid washs with ammonia solution, discards ammonia solution, evaporate to dryness, residue add dissolve with methanol and are transferred in the volumetric flask, are diluted to scale with methyl alcohol, shake up, filter with miillpore filter (0.45 μ m), promptly.

Method 4: the product of getting it filled, porphyrize, the accurate title, decide, and puts in the apparatus,Soxhlet's, adds methyl alcohol, and placement is spent the night, and it is an amount of to add methyl alcohol again, reflux, extract reclaims solvent and evaporate to dryness, and residue adds water, and low-grade fever makes dissolving, and water liquid extracts with water-saturated n-butanol; Normal butyl alcohol liquid washs with ammonia solution, discards ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, and residue adds water, low-grade fever makes dissolving, put cold, by D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), the water wash-out discards water liquid, uses 30% ethanol elution again, discards eluent, continue and use 70% ethanol elution, collect eluent, evaporate to dryness, residue adds methyl alcohol makes dissolving, and quantitatively is transferred in the measuring bottle, is diluted to scale with methyl alcohol, shake up, filter with miillpore filter (0.45 μ m), promptly.

Method 5: the product of getting it filled, porphyrize accurate claims surely, puts in the apparatus,Soxhlet's, adds 2% potassium hydroxide methanol solution, reflux, extract evaporate to dryness, residue add and are transferred in the separating funnel after water makes dissolving, use ethyl acetate extraction; Acetic acid ethyl fluid adds water washing; Merge water liquid, extract with water-saturated n-butanol; N-butanol extracting liquid washs with ammonia solution, discards ammoniacal liquor, normal butyl alcohol liquid evaporate to dryness, residue adds water makes dissolving, put cold, by D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), the water wash-out discards water liquid, use 70% ethanol elution again, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in the measuring bottle, add methanol constant volume to scale, shake up, filter with miillpore filter (0.45 μ m), promptly.

Sample	Method 1 (mg/g)	Method 2 (mg/g)	Method 3 (mg/g)	Method 4 (mg/g)	Method 5 (mg/g)
						1	0.3217	0.3312	0.3252	0.3856	0.4193
2	0.3262	0.3442	0.3321	0.3791	0.4156
						3	0.3305	0.3361	0.3289	0.3903	0.4166

According to test findings as can be known, employing method 5 preparation need testing solutions, Astragaloside IV extracts fully, and is easy and simple to handle, and the extraction ratio of method one, method two and method three is lower, residue is more, bad operation when shifting constant volume, and the impurity of sample disturbs bigger simultaneously, method four content when washing with 30% ethanol has loss, after relatively methodological study is carried out in system of selection five, confirmed the reliability of this method, so select this kind method.

2, the investigation of extraction time

6 parts of sample thiefs take by weighing about 1g respectively, and accurate the title decides, put in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution 70ml, per two parts of Soxhlet extraction time was respectively 3 hours, 4 hours, 6 hours, according to preparation method's preparation of need testing solution under the Radix Astragali assay item in the method for quality control of the present invention, by measuring, the result shows that to extract 4 hours very approaching with 6 hours result, and 3 hours result differs greatly, thus the selective extraction time be 4-6 hour, can extract Astragaloside IV fully.See the following form.

The investigation of extraction time

3, the investigation of normal butyl alcohol extraction time

6 parts of sample thiefs, take by weighing about 1g respectively, the accurate title, decide, preparation method's preparation (extracting respectively 2 times, 3 times, 4 times, 5 times) according to need testing solution under the Radix Astragali assay item in the method for quality control of the present invention with water saturated normal butyl alcohol, by measuring, the result shows extraction for the second time not exclusively, and difference is very big, also have a spot of Astragaloside IV in the n-butanol extracting liquid of the 4th, and Astragaloside IV has been less than 0.01% in the normal butyl alcohol liquid of the 5th.Therefore the extraction time with normal butyl alcohol is decided to be 3-5 time, and optimum extraction time is 4 times.See the following form.

The investigation of extraction time

(2) selection of moving phase:

Moving phase 1: the mixed solution with methyl alcohol, ethyl acetate solution different proportion is a moving phase.

Moving phase 2: the mixed solution with first alcohol and water different proportion is a moving phase.

Moving phase 3: the mixed solution with acetonitrile and water different proportion is a moving phase.

The result: with acetonitrile: water=10-90: 90-10 is moving phase; The negative sample chromatogram is at non-false positive peak, Astragaloside IV position, and Astragaloside IV separates fully (degree of separation＞1.5) with close impurity peaks, and promptly Astragaloside IV and other component peaks reach baseline separation under this condition.Optimal flow is mutually: acetonitrile: water=36: 64.

(3) investigation of linear relationship

In the measuring bottle of accurate absorption reference substance storing solution 1ml to 10ml, 1ml to 5ml, 10ml to 25ml, 3ml to 5ml, add the methyl alcohol dilution and be settled to scale, shake up, get final product.Accurate respectively then above-mentioned reference substance solution and each 10 μ l injection liquid chromatograph of stock solution drawn, measure its peak area, and be ordinate with the natural logarithm (LNA) of peak area value A, the natural logarithm of sample size C (LNC) is a horizontal ordinate, getting regression equation is: LNA=1.5525LNC+16.324, r=0.9994.Natural logarithm (LNA) with peak area A is mapped to the natural logarithm (LNC) of sample size C, gets a straight line, and its result shows that the linear relationship of Astragaloside IV is good in 0.4064～4.064 μ g scope.See the following form.

The Astragaloside IV linear relationship is investigated

(4) replica test:

Get 6 parts in same sample, every part of about 1g, the accurate title, decide, according to preparation method's preparation of need testing solution under the Radix Astragali assay item in the method for quality control of the present invention, accurate respectively absorption reference substance solution 5 μ l and 10 μ l, need testing solution 10 μ l sample introductions, record chromatogram, calculate content, it is 0.4118mg/g that the Astragaloside content of 6 duplicate samples is measured mean value, and RSD is 0.5%, illustrates that repeatability is good.See the following form.

Replica test

(5) recovery test:

Get 6 parts in the sample (average content 0.4118mg/g) of known content, every part of about 0.5g, the accurate title, decide, put in the apparatus,Soxhlet's, accurate respectively Astragaloside IV reference substance solution (0.2392mg/ml) 1ml that adds, according to preparation method's preparation of need testing solution under the Radix Astragali assay item in the method for quality control of the present invention, make application of sample and reclaim need testing solution.Measure Astragaloside content, recording average average recovery is 97.86%, and RSD is 1.1%, and the result shows that this method has good average recovery.See the following form.

Recovery test

(6) reappearance test

The product of getting it filled, take a sample according to preparation method's preparation of need testing solution under the Radix Astragali assay item in the method for quality control of the present invention by the different people of group respectively, accurate respectively absorption reference substance solution 5 μ l and 10 μ l, need testing solution 10 μ l sample introductions, the record chromatogram, calculate content, the Astragaloside content mean value of three parts of medicines is respectively 0.4203mg/g, 0.4127mg/g, 0.3946mg/g, and RSD% is respectively 0.3%, 0.3%, 0.2%, illustrates that reappearance is good.See the following form.

The reappearance test

(7) scope is investigated

According to the content limit of Astragaloside IV in the medicine, its content is less than 1%, and the scope of investigation is ± 50%.The preparation of test sample is according to preparation method's preparation of need testing solution under the Radix Astragali assay item in the method for quality control of the present invention, and the accurate respectively 10 μ l of absorption inject liquid chromatograph, and the record peak area calculates content.See the following form.

Scope is investigated

Measurement result shows, can reach requirement in two extreme its test results of scope.

(8) serviceability test

1. stability experiment product of getting it filled prepare test liquid by the preparation method of test liquid under the Radix Astragali assay item in the method for quality control of the present invention, and respectively at 0,2,4,6,8,12h is the peak area value of Astragaloside IV in the working sample respectively.RSD% is 0.2%, and the result shows that sample solution is stable in 12h.

Stability experiment

2. different chromatographic columns are relatively measured its content with same sample and are compared under different chromatographic columns.See the following form.

Different chromatographic columns

3. the variation of moving phase proportion of composing relatively compares same sample its content of mensuration under difference flows the phase composition ratio.See the following form.

The different phase composition ratios that flow

4. different column temperatures are relatively measured its content with same sample and are compared under different column temperatures.See the following form.

Different column temperatures

(9) sample determination prepares 10 batches of totally 20 duplicate samples with the preparation method of need testing solution under the Radix Astragali assay item in the method for quality control of the present invention, surveys its Astragaloside content.See the following form.

Astragaloside IV determination test in the sample

Two, the research of cantharidin content assaying method

Because of cantharidin is anticancer effective ingredient in the preparation of the present invention, and a lot of flavour of a drug are directly to be used as medicine with the medicinal material fine powder in the preparation of the present invention, and with general backflow, ultrasonic Extraction the time, impurity is more, for cantharidin in the preparation being extracted fully and removing too much impurity, it has been carried out methodological study.

(1) need testing solution preparation method research

1, determining of extracting method:

Method one: the thing porphyrize of getting it filled, the accurate title, decide, and puts in the round-bottomed flask, add methenyl choloride, put in the water-bath and reflux, put cold, filter, residue adds methenyl choloride to be continued to reflux, and puts cold, filter, merging filtrate is put in the water-bath and is volatilized, residue dissolves with methenyl choloride, transfer is settled in the volumetric flask, shakes up, promptly.

Method two: the thing porphyrize of getting it filled, the accurate title, decide, and puts in the round-bottomed flask, adds methenyl choloride, put in the water-bath and reflux, put coldly, filter, residue adds a small amount of methenyl choloride washing, and merging filtrate volatilizes naturally, with sodium chloride-sodium hydroxide solution washing residue, transfer pH to 1～2 with hydrochloric acid again, filter, filtrate is used chloroform extraction, and chloroform solution volatilizes naturally, and residue adds the methenyl choloride dissolving, and shift quantitatively to measuring bottle, shake up, promptly.

Method three: the thing porphyrize of getting it filled, the accurate title, decide, and puts in the beaker, add sodium chloride-sodium hydroxide solution, sonicated is put cold, transfer pH to 1～2 with hydrochloric acid, suction filtration washs residue with low amounts of water, merging filtrate is used chloroform extraction, and chloroform solution volatilizes naturally, residue adds the methenyl choloride dissolving, and shift quantitatively to measuring bottle, shake up, promptly.

Method four: the thing porphyrize of getting it filled, the accurate title, decide, and puts in the beaker, adds sodium chloride-sodium hydroxide solution, ultrasonic, put coldly, add concentrated hydrochloric acid, stir, leave standstill, put coldly, suction filtration washs residue with low amounts of water, merging filtrate, with chloroform extraction (if when in leaching process, emulsion occurring, in order not lose cantharidin content, must be after having extracted that emulsion layer is centrifugal, divide and get chloroform solution), combined chloroform liquid is put 50 ℃ of water-baths and is waved to about 1～2ml, be settled to 3ml with chloroform again, shake up, promptly.

The result: the test of many times by above-mentioned four kinds of methods compares, and the impurity that method one is extracted is more; The method two poor reproducibility, extraction ratio is also lower simultaneously; Impurity is few after treatment with method four samples for method three, and there are two problems in the extraction ratio height but method three volatilizes the back naturally at chloroform extracted solution: the first, after chloroform volatilizes naturally, can separate out low amounts of water, and cause constant volume inaccurate, influence measurement result; The second, after chloroform volatilizes naturally, can cause the cantharidin content loss simultaneously, cause poor reproducibility.And in method four, sample is just can overcome above two problems with 50 ℃ of water-bath Back stroke to about 1～2ml, through test of many times, and favorable reproducibility.So the test sample extraction scheme is chosen to be method four.

2, extract determining of solvent volume

The thing of getting it filled, preparation method's preparation according to need testing solution under the cantharidin assay item in the method for quality control of the present invention, relatively extract solvent sodium chloride sodium hydroxide solution and (get 1mol/L sodium hydroxide solution 100mL and sodium chloride 2g, stir dissolving) be three kinds of methods of 20ml, 30ml, 50ml.See the following form.

Extract the comparison of solvent volume

Solvent load (ml)	20	30	50
				Content (μ g/g)	51.22	55.14	55.29

By the comparison of test findings, solvent volume can be extracted cantharidin fully when 30ml, 50ml, is the extraction solvent of 30-50ml so adopt volume, preferred 30ml.

3, extraction time determines

The thing of getting it filled is according to preparation method's preparation of need testing solution under the cantharidin assay item in the method for quality control of the present invention, more ultrasonic 20min, 30min, 40min, four kinds of methods of 60min.See the following form.

The comparison of extraction time

Ultrasonic time (min)	20	30	40	60
					Content (mg/g)	52.31	54.94	54.28	53.75

By the comparison of test findings, ultrasonic time can extract cantharidin in the time of 30-40 minute fully, so adopt ultrasonic 30-40min, preferred 30min.

4, the evaporation water bath temperature determines

The thing of getting it filled according to preparation method's preparation of need testing solution under the cantharidin assay item in the method for quality control of the present invention, is relatively used 40 ℃, and 50 ℃, 55 ℃, four kinds of methods of the 60 ℃ of chloroform solution to 1 that volatilizees～2ml.See the following form.

The comparison of evaporation water bath temperature

Bath temperature (℃)	40	50	55	60
					Content (mg/g)	55.37	55.15	54.92	52.76

By the comparison of test findings, prove with 50 ℃ of water-baths chloroform that volatilizees and neither lose cantharidin content, moisture content can be flung to again, so the present invention adopts 50 ℃ of water-baths chloroform that volatilizees.

5, extract determining of solvent

The thing of getting it filled, according to preparation method's preparation of need testing solution under the cantharidin assay item in the method for quality control of the present invention, relatively extracting solvent is chloroform and two kinds of methods of methylene chloride.See the following form.

Extract the comparison of solvent volume

Solvent	Chloroform	Methylene chloride
			Content (μ g/g)	54.97	48.69

By the comparison test result, prove that chloroform can extract cantharidin fully, and the methylene chloride extraction ratio is lower.So it is to extract solvent that the present invention adopts chloroform.

(2) specificity test

The preparation of need testing solution: according to preparation method's preparation of need testing solution under the cantharidin assay item in the method for quality control of the present invention.

The preparation of negative sample solution: get not cantharidal negative sample, prepare with the need testing solution preparation method.

Under selected chromatographic condition, draw cantharidin reference substance solution, need testing solution and scarce Chinese blister beetle negative sample solution 2 μ l inject gas chromatographs respectively.The retention time of cantharidin is about 12min, and negative sample is at noiseless peak, reference substance position, and cantharidin peak and other impurity peaks degree of separation are all greater than 1.5 in the sample, and theoretical cam curve is pressed the cantharidin peak and calculated greater than 1000.

(3) investigation of linear relationship

Precision takes by weighing cantharidin reference substance 12.10mg, puts in the 50ml volumetric flask, adds chloroform dissolving and dilution and is settled to scale, shakes up, in contrast the product stock solution.Accurate respectively again absorption reference substance stock solution is an amount of, and chlorination is copied into the reference substance solution of 0.121mg/ml, 0.0605mg/ml, 0.0484mg/ml, 0.0242mg/ml, 0.0121mg/ml.Accurate respectively then each the 2 μ l inject gas chromatograph of above-mentioned reference substance solution of drawing are measured its peak area, and with peak area value (A) sample size (C) are returned, and get the typical curve equation and are: A=6127836.8097C+4767.9444, r=0.9998; Match to former point equation is: A=6142992.3864C, r=0.9998.The result shows that the cantharidin linear relationship is good in 0.0242 μ g～0.484 μ g scope.See the following form.

The cantharidin linear relationship is investigated

With two Equation for Calculating in the substitution of same sample peak area (577921) difference, the RSD% of income value is 0.3%, therefore can calculate with one point external standard method.

(4) replica test

Sample thief, precision takes by weighing 6 parts respectively, every part of about 2g, according to preparation method's preparation of need testing solution under the cantharidin assay item in the method for quality control of the present invention, the accurate respectively 2 μ l sample introductions of drawing, record chromatogram, calculate content, 6 duplicate samples cantharidin assay mean values are 55.03 μ g/g, and RSD% is 1.63%, illustrate that repeatability is good.See the following form.

Replica test

(5) accuracy test

The test of employing average recovery.Get 6 parts in the sample of known content, every part of about 1g, the accurate title, decide, put in the 100ml beaker, add sodium chloride sodium hydroxide solution (get 1mol/L sodium hydroxide solution 100mL and sodium chloride 2g, stir, dissolving) 30ml respectively, respectively accurate again cantharidin reference substance solution (the 48.4 μ g/ml) 1ml that adds prepares recovery need testing solution according to the preparation method of need testing solution under the cantharidin assay item in the method for quality control of the present invention.Because the cantharidin reference substance solution is to use chloroform as solvent, because of chloroform and sodium chloride-NaOH mixed solution do not dissolve each other, cause having not ease for operation in extraction and the suction filtration step, therefore in the average recovery test, use the preparation of cantharidin reference substance solution instead acetone as solvent.Through relatively, be the cantharidin reference substance peak area basically identical of solvent preparation with chloroform or acetone.

The preparation of reference substance solution: precision takes by weighing cantharidin reference substance 0.01210g in the 50ml measuring bottle, adds acetone solution and is diluted to scale, shakes up.Precision is measured 10ml in the 50ml measuring bottle again, adds acetone diluted to scale, shakes up, and promptly gets (C=48.4 μ g/ml).

Test method: 6 parts in sample getting known content, every part of about 1g, the accurate title, decide, put in the 100ml beaker, add the sodium chloride sodium hydroxide solution respectively and (get 1mol/L sodium hydroxide solution 100mL and sodium chloride 2g, stir, dissolving) 30ml, accurate respectively again cantharidin reference substance solution (C=48.4 μ g/ml, acetone the are solvent) 1ml that adds, preparation method according to need testing solution under the cantharidin assay item in the method for quality control of the present invention prepares recovery need testing solution, the accurate respectively 2 μ l sample introductions of drawing, the record chromatogram calculates content, recording average average recovery is 100.45%, and RSD% is 2.37%.The result shows that this method has good average recovery.See the following form.

Recovery test

(6) reappearance test

Sample thief, shine the preparation method of need testing solution preparation under the cantharidin assay item in the method for quality control of the present invention in the laboratory by different people respectively, the accurate respectively 2 μ L sample introductions of drawing, the record chromatogram, calculate content, the content mean value of three duplicate samples is respectively 77.74 μ g/g, 52.92 μ g/g, 55.80 μ g/g, and RSD% is respectively 1.81%, 2.03%, 0.22%, illustrates that reappearance is good.See the following form.

The reappearance test

(7) scope is investigated

The content limit of cantharidin in according to the present invention, its content are less than 1%, and the scope of investigation is ± 50%.Sample thief, precision takes by weighing 4 parts respectively, two parts of about 1g, two parts of about 3g, according to preparation method's preparation of need testing solution under the cantharidin assay item in the method for quality control of the present invention, the accurate respectively 2 μ l sample introductions of drawing, the record chromatogram, calculate content, 4 duplicate samples cantharidin assay mean values are 54.09 μ g/g, and RSD is 1.91%, with repeated sample size (55.03 μ g/g) relative standard deviation be 1.22%, declared range test is good.See the following form.

Scope is investigated

(8) serviceability test

1. stability experiment

Get compound cantharidin oral preparations, prepare need testing solution according to the preparation method of need testing solution under the cantharidin assay item in the method for quality control of the present invention, respectively at 0,2,4,8, accurate 2 μ L inject gas chromatographs, the cantharidin peak area value in the working sample drawn of 12h.RSD% is 0.3%, and the result shows that sample solution is stable in 12h.See the following form.

Stability experiment

2. different gas chromatographs, different chromatographic column are relatively measured its content with same sample and are compared under different chromatographic columns.It is 55.80mg/g that two kinds of different conditions record content mean value, and RSD% is 0.22%.See the following form.

Different chromatographic columns

Annotate: " A " is: chromatographic column: with carbowax-20M and methyl silicone rubber (SE-30) is immobile liquid, and coating concentration is respectively 10% and 5%, 1: 1 mixes the dress post; Ultrapure nitrogen (55KPa), hydrogen (50ml/min), air (500ml/min); Column temperature: 155 ℃; Injector temperature: 240 ℃; Fid detector, detector temperature: 240 ℃; Weil-McLain jade for asking rain workstation;

" B " is: chromatographic column: with polyphenyl methyl siloxane (5% phenyl) capillary column (30m * 0.25mm * 0.25 μ m); N ₂Be carrier gas, linear velocity 24mL/min; Split ratio 10: 1; Hydrogen flowing quantity: 40.0ml/min; Air mass flow: 400.0ml/min; 135 ℃ of column temperatures; 240 ℃ of vaporizer temperature; Fid detector, 240 ℃ of detector temperatures; The GCSolution workstation.

(9) sample determination

Preparation method with need testing solution under the cantharidin assay item in the method for quality control of the present invention prepares 12 batches, and totally 24 duplicate samples are surveyed its cantharidin content.See the following form.

The sample determination test

Three, genseng, Radix Astragali thin layer Study on Identification

Differentiate genseng, Milkvetch Root in the preparation with ginsenoside Rg1's reference substance and Astragaloside IV reference substance:

Need testing solution preparation method one: the product of getting it filled, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put refluxing extraction in the water-bath, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put refluxing extraction in the water-bath, reclaim methyl alcohol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separating funnel, wash container again with water, be transferred in the separating funnel, extract with water saturated normal butyl alcohol, extract washes with water, divides and gets normal butyl alcohol liquid, evaporate to dryness, the dissolving of residue water is transferred to (neutral alumina 100～200 orders, 2g on the alumina column, dry column-packing, diameter 1cm, long 25cm), use earlier the chloroform wash-out, discard the chloroform eluent, use methanol-eluted fractions again, eluent is evaporate to dryness in water-bath, residue adds methyl alcohol makes dissolving, as need testing solution; The negative test liquid that is equipped with the shortage of staff's ginseng and the Radix Astragali with legal system.

Need testing solution preparation method two: the product of getting it filled, porphyrize adds an amount of chloroform, ultrasonic Extraction discards chloroform solution, treats that chloroform waves to the greatest extent, add an amount of methyl alcohol, ultrasonic Extraction reclaims methyl alcohol to doing, and residue hydro-oxidation sodium solution makes dissolving, go in the separating funnel, wash container again with water, be transferred in the separating funnel, extract with water saturated normal butyl alcohol, extract washes with water, divide and get normal butyl alcohol liquid, evaporate to dryness, the dissolving of residue water, be transferred to and use the chloroform wash-out on the alumina column, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol makes dissolving, as need testing solution; The negative test liquid that is equipped with the shortage of staff's ginseng and the Radix Astragali with legal system.

Developping agent is selected: respectively with the mixed solution of chloroform, ethyl acetate, methyl alcohol, water different proportion; The mixed solution of methenyl choloride, methyl alcohol, water different proportion; The mixed solution of chloroform, ethyl acetate, water different proportion is a developping agent.

The thickness of thin layer plate is selected: more than the thickness 500 μ m; Below the thickness 500 μ m.

The point sample amount is selected: be test sample 8 μ l with the point sample amount respectively, and reference substance 4 μ l; The point sample amount is test sample 10 μ l, and reference substance 5 μ l select.

The result: employing method one preparation need testing solution, with methenyl choloride: methyl alcohol: water=5-50: 2-20: 0.5-10 is a developping agent, and its degree of separation is good, and the spot colour developing is clear, and negative control is noiseless, the method favorable reproducibility.With chloroform: ethyl acetate: methyl alcohol: water is developping agent, and standing time is longer, operation inconvenience; And if the humidity of operating environment more also can cause the spot separating effect bad, reappearance is relatively poor, by test of many times, think the thin layer plate behind the point sample to be placed the baking oven about 65 ℃ to heat 10～15min or place the expansion cylinder opposite side to come controlled humidity with 5ml sulfuric acid; The thickness of thin layer plate also is a key factor simultaneously, and through test of many times, this discriminating needs just can reach good expansion effect with slab (more than the 500 μ m); Be to control medicinal material content with the total amount of genseng Re and Rg1 greater than 0.30% in the genseng, adopting the point sample amount is test sample 8 μ l, and the spot that content junior in the two can appear in reference substance 4 μ l is fuzzy; In addition, the test sample treatment step is more, also easily causes damage.So best developping agent is: methenyl choloride: methyl alcohol: water=13: 7: 2, more than the thickness 500 μ m of thin layer plate, the point sample amount is test sample 10 μ l, during reference substance 5 μ l, identification result the best.

Four, Sculellaria barbata thin layer Study on Identification

Differentiate Sculellaria barbata medicinal material in the preparation with the Sculellaria barbata reference substance:

Need testing solution preparation method one: the product of getting it filled, porphyrize adds diethyl ether, and reflux is put coldly, filters, and discards filtrate, and the dregs of a decoction add methyl alcohol, and reflux is put coldly, filters, and filtrate evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; The negative test liquid that lacks Sculellaria barbata with the method preparation.

Need testing solution preparation method two: the product of getting it filled, porphyrize adds methenyl choloride, and ultrasonic Extraction filters, and discards filtrate, and the dregs of a decoction add methyl alcohol, and ultrasonic Extraction filters, and filtrate evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; The negative test liquid that lacks Sculellaria barbata with the method preparation.

Developping agent is selected: respectively with the mixed solution of toluene, ethyl formate, formic acid different proportion; The mixed solution of cyclohexane, ethyl acetate different proportion; The mixed solution of benzene and ethyl formate different proportion; The mixed solution of cyclohexane, formic acid different proportion is a developping agent.

The result: employing method one preparation need testing solution, with toluene: ethyl formate: formic acid=2-20: 1-9: 1-9 is a developping agent, and its degree of separation is good, and the spot colour developing is clear, and negative control is noiseless, the method favorable reproducibility, specificity is strong.Best developping agent is: toluene: ethyl formate: formic acid=9: 5: 3.

Five, fruit of medicinal cornel thin layer Study on Identification

Method one: the thing of getting it filled, porphyrize adds ethyl acetate, and sonicated filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol makes dissolving, as need testing solution; The negative test liquid that lacks the fruit of medicinal cornel with the method preparation.Other gets the ursolic acid reference substance and adds solution that absolute ethyl alcohol is mixed with 1mg/ml product solution in contrast.With toluene: ethyl acetate: formic acid=20: 4: 0.5 is developping agent, launches, and takes out, and dries, and the ethanol solution of sulfuric acid with 10% is a developer.

The result: negative sample has interference.

Method two: the thing of getting it filled, porphyrize adds methyl alcohol, and reflux filters, and discards filtrate.Residue adds ethyl acetate, and sonicated filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol makes dissolving, as need testing solution; The negative test liquid that lacks the fruit of medicinal cornel with the method preparation.Get fruit of medicinal cornel control medicinal material, be equipped with control medicinal material solution with legal system.With cyclohexane: chloroform: ethyl acetate=20: 5: 8 is developping agent, launches, and takes out, and dries, and the ethanol solution of sulfuric acid with 10% is a developer.

The result: negative sample has interference.

Method three: the thing of getting it filled, porphyrize adds methyl alcohol, and sonicated filters, the filtrate evaporate to dryness, residue adds absolute ethyl alcohol: the mixed solution of chloroform=3: 2 makes dissolving, as need testing solution; The negative test liquid that lacks the fruit of medicinal cornel with the method preparation.Other gets fruit of medicinal cornel control medicinal material, is equipped with control medicinal material solution with legal system.With cyclohexane: acetone: ethyl acetate=10: 4: 2 is developping agent, launches, and takes out, and dries, and the ethanol solution of sulfuric acid with 10% is a developer.

The result: negative sample has interference.

Owing to all contain ursolic acid and oleanolic acid in the fruit of medicinal cornel and the fruit of glossy privet, can cause negative the interference, therefore consideration selects for use in the fruit of medicinal cornel distinctive loganin to differentiate.

Differentiate fruit of medicinal cornel medicinal material in the preparation with the loganin reference substance:

Need testing solution preparation method one: the thing of getting it filled, grind well, add 50% methyl alcohol, reflux is put coldly, filters, filtrate evaporate to dryness, residue add 50% methyl alcohol and dissolve in right amount, be added on the neutral alumina post (100～200 orders, 4g) on, use 40% methanol-eluted fractions.Collect eluent, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; The negative test liquid that lacks the fruit of medicinal cornel with the method preparation.

Need testing solution preparation method two: the thing of getting it filled, grind well, add 50% methyl alcohol, sonicated is put coldly, filters, filtrate evaporate to dryness, residue add 50% methyl alcohol and dissolve in right amount, be added on the neutral alumina post (100～200 orders, 4g) on, use 40% methanol-eluted fractions.Collect eluent, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; The negative sample solution that lacks the fruit of medicinal cornel with the method preparation.

Developping agent is selected: respectively with the mixed solution of toluene, ethyl acetate, formic acid different proportion;

The mixed solution of toluene, acetone, absolute ethyl alcohol, dense ammonia different proportion;

The mixed solution of toluene, acetone, absolute ethyl alcohol, formic acid different proportion;

The mixed solution of cyclohexane, acetone, ethyl acetate different proportion;

The mixed solution of cyclohexane, chloroform, ethyl acetate different proportion is a developping agent.

The result: employing method one preparation need testing solution, with toluene: acetone: absolute ethyl alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is a developping agent, and its degree of separation is good, and the spot colour developing is clear, and negative control is noiseless, the method favorable reproducibility.In addition, the point sample shape is the effective of round dot for band shape than point sample shape.Best developping agent is: toluene: acetone: absolute ethyl alcohol: formic acid=4: 16: 2: 0.5.Adopt first three methods that the fruit of medicinal cornel is carried out thin layer and differentiate that its identification result degree of separation is bad, the spot colour developing is unintelligible, and feminine gender has interference.

Six, fruit of glossy privet thin layer Study on Identification

Differentiate fruit of glossy privet medicinal material in the preparation with fruit of glossy privet control medicinal material:

Need testing solution preparation method one: the thing of getting it filled, grind well, add methyl alcohol, reflux is put coldly, filters, the filtrate evaporate to dryness, residue adds absolute ethyl alcohol: the mixed solution of methenyl choloride=3: 2 makes dissolving, as need testing solution; The negative test liquid that lacks the fruit of glossy privet with the method preparation.

Need testing solution preparation method two: the thing of getting it filled, porphyrize, boiling filters, and filtrate concentrates, and transfers pH to 2～3 with watery hydrochloric acid, with ethyl acetate extraction, combined ethyl acetate liquid, evaporate to dryness, residue add ethyl acetate makes dissolving, as need testing solution; The negative test liquid that lacks the fruit of glossy privet with the method preparation.

Need testing solution preparation method three: the thing of getting it filled, porphyrize, boiling filters the filtrate evaporate to dryness.Residue adds absolute ethyl alcohol: the mixed solution of methenyl choloride=3: 2 makes dissolving, as need testing solution; The negative test liquid that lacks the fruit of glossy privet with the method preparation.

Need testing solution preparation method four: the thing of getting it filled, porphyrize adds the methyl alcohol reflux, filters, and discards filtrate, and residue adds ethyl acetate, and is ultrasonic, filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol makes dissolving, as need testing solution; The negative test liquid that lacks the fruit of glossy privet with the method preparation.

Need testing solution preparation method five: the thing of getting it filled, porphyrize adds ethyl acetate, and is ultrasonic, filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol makes dissolving, as need testing solution; Lack the negative test liquid of the fruit of glossy privet with the method preparation.

Developping agent is selected: respectively with the mixed solution of cyclohexane, acetone, ethyl acetate different proportion;

The mixed solution of toluene, ethyl acetate, formic acid different proportion;

The mixed solution of sherwood oil, ethyl acetate, formic acid different proportion is a developping agent.

The result: the negative sample of preceding four kinds of methods has interference.The sample effect that method five is handled is better, and is negative noiseless.So final employing method five preparation need testing solutions, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is a developping agent, and its degree of separation is good, and the spot colour developing is clear, and negative control is noiseless, and the method favorable reproducibility differentiates that specificity is strong.Best developping agent is: toluene: ethyl acetate: formic acid=20: 4: 0.5.

Seven, the thin layer Study on Identification of bear gall powder

Bear gall powder ought to be controlled for the valuable medicinal in the prescription, and the spy makes following thin layer Study on Identification to it:

Method one: the thing of getting it filled, add 10% sodium hydroxide solution, add a cover little boiling.Put coldly, filter, filtrate drips watery hydrochloric acid to pH 2～3, goes in the separating funnel, use ethyl acetate extraction, the merging extract, and evaporate to dryness, residue add ethanol makes dissolving, as need testing solution; The negative test liquid that lacks bear gall powder with the method preparation.

Method two: the thing of getting it filled, add 10% sodium hydroxide solution, put little boiling on the electric furnace.Put coldly, filter, filtrate drips watery hydrochloric acid to pH 2～3, goes in the separating funnel, use ethyl acetate extraction, the merging extract, and evaporate to dryness, residue add ethanol makes dissolving, as need testing solution; The negative test liquid that lacks bear gall powder with the method preparation.

Method three: the thing of getting it filled adds 10% NaOH, ultrasonic dissolving, 120 ℃ of heating hydrolysis of making.Put coldly, filter, filtrate drips watery hydrochloric acid to pH 2～3, goes in the separating funnel, with ethyl acetate extraction twice, merges extract, and evaporate to dryness, residue add ethanol makes dissolving, as need testing solution; The negative test liquid that lacks bear gall powder with the method preparation.

Method four: the thing of getting it filled, add the water heated and stirred it is fully dissolved, leave standstill, put coldly, filter.Filtrate is extracted with the ether jolting, discards ether layer, and water layer adds 20% NaOH in water-bath heating hydrolysis (replenishing the water of evaporation at any time).Put coldly, drip watery hydrochloric acid, go in the separating funnel, use ethyl acetate extraction to pH 2～3, the merging extract, evaporate to dryness, residue add ethanol makes dissolving, as need testing solution; The negative test liquid that lacks bear gall powder with the method preparation.

Method five: the thing of getting it filled, add the ethanol jolting and extract, merge ethanol extract, filter, filtrate is put evaporate to dryness in the water-bath.Residue adds 20% NaOH and is transferred in the appropriate containers, hydrolysis to the water-bath.Put coldly, dripping hydrochloric acid goes in the separating funnel to PH 2～3, uses ethyl acetate extraction, merges extract, and evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; The negative test liquid that lacks bear gall powder with the method preparation.

Developping agent is selected: respectively with the mixed solution of isooctane, ether, glacial acetic acid, normal butyl alcohol, water different proportion; The mixed solution of isooctane, ethyl acetate, glacial acetic acid different proportion is a developping agent.

Result: respectively with 10% ethanol solution of sulfuric acid, 10% phosphomolybdic acid ethanol solution colour developing, with the corresponding position of reference substance (urso, chenodeoxycholic acid, hyodesoxycholic acid and cholic acid mix reference substance) on, spot is unintelligible, and other impurity colors are very dark, disturbs bigger.Observe under fluorescence, impurity is also very big to main spot influence.

The discrimination method of bear gall powder in the capsule is answered in reference ten thousand.The sampling amount of its test sample is 1.8g, converts by the amount to bear gall powder in this preparation, and the sampling amount that draws oral formulations of the present invention is about 20-25g, and sampling amount is excessive, and sample preparation process complexity, easily causes damage; In addition, oral formulations taste of traditional Chinese medicine of the present invention is more, has 11 flavors, can cause larger interference, so tool operability not, can't formulate discrimination method to bear gall powder.

Eight, the inspection method research of chloroform residual quantity

The Chinese blister beetle medicinal material is to use the chloroform lixiviate in extraction process among the present invention, and chloroform toxicity is bigger, being two kind solvents that country lists, is to be strict with its limit of control in medicine, and the chloroform residual quantity in two appendix of 2005 editions pharmacopeia in the regulation medicine should be controlled at 0.006%.The applicant has carried out the methodology checking to the inspection of chloroform residual quantity in the preparation of the present invention for this reason, and this method specificity is strong, highly sensitive, can effectively control the chloroform residual quantity in the preparation.

(1) instrument and reagent

Tianjin, instrument island GC-2010 gas chromatograph; Fid detector; RESTEC Rtx-5 quartz capillary gas chromatographic column (30m * 0.25mm * 0.25 μ m); The GC-Solution workstation; The GCH-300 hydrogen generator; GCB-2000 full-automatic air source; DANI HSS 86.50 headspace sampling devices; The AE200 analytical balance.

Reagent and reagent chloroform are chromatographically pure, N, and dinethylformamide (DMF) is pure for analyzing.

(2) specificity test

A. chromatographic condition: chromatographic column: fused-silica capillary column (column length 30m, internal diameter 0.25mm, film thickness 0.25 μ m) Rtx-5 (5% phenyl methyl siloxane is a stationary phase); Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Carrier gas: high purity nitrogen; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min.Adopt headspace sampling: the vaporizer temperature is 110 ℃, heating 30min, 115 ℃ of sample introduction needle temperature, 120 ℃ of transfer tube temperature.

B. the head space condition is selected: with 75%N, when dinethylformamide (DMF) is solvent, lower temperature can not make chloroform reach fully saturated, it is lower to detect sensitivity, respectively furnace temperature is set at 90 ℃, 100 ℃, 110 ℃ and 120 ℃, balance 30min, the result shows, to detect peak area the highest for identical reference substance in the time of 110 ℃, and 90 ℃ to detect peak area minimum, so select 110 ℃ of best furnace temperature of conduct.Also compared simultaneously the difference between balance 20min, 30min, 45min, the 60min under the identical furnace temperature, the result shows that balance 20min can not reach fully saturated, gas leakage then may take place because equilibration time is long in 45min and 60min, because 30min peak area maximum, so final selection balance Best Times is 30min.

C. specificity test: accurate reference substance solution, need testing solution and each 2ml of negative sample of drawing rolls lid in 20ml head space bottle, sample introduction behind 110 ℃ of balance 30min in the head space stove, record chromatogram.From chromatogram as can be seen, the retention time at chloroform peak is 3.121min, all reaches fully with other peak in solvent and the negative sample and separates, and degree of separation is greater than 2.0, the theoretical cam curve at chloroform peak is 112150, should be not less than 5000 so determine number of theoretical plate with the calculating of chloroform peak.

(3) preparation of reference substance solution: it is an amount of to get the chloroform reference substance, adds 75%N, and dinethylformamide (DMF) is made the solution that every 1ml contains 15 μ g, promptly.

(4) preparation of need testing solution:

Method one: sample thief is in 20ml head space bottle, and accurate the title decides, and precision adds entry, rolls lid, and jolting is even, promptly.

Method two: sample thief is in tool plug test tube, and accurate the title decides, and precision adds entry, close plug, and jolting, centrifugal, precision is measured supernatant in 20ml head space bottle, rolls lid, promptly.

Method three: sample thief is in tool plug test tube, and accurate the title decides, the accurate 50%N that adds, and dinethylformamide (DMF), close plug, jolting, centrifugal, precision is measured supernatant in 20ml head space bottle, rolls lid, promptly.

Method four: sample thief is in tool plug test tube, and accurate the title decides, the accurate 75%N that adds, and dinethylformamide (DMF), close plug, jolting, centrifugal, precision is measured supernatant in 20ml head space bottle, rolls lid, promptly.

Method five: sample thief is in tool plug test tube, and accurate the title decides, the accurate N that adds, and dinethylformamide (DMF), close plug, jolting, centrifugal, precision is measured supernatant in 20ml head space bottle, rolls lid, promptly.

When handling sample by above five kinds of methods, reference substance is also prepared with corresponding solvent, and has carried out recovery test.By above five kinds of methods, recovery test is only with 75%N, dinethylformamide (DMF) and N, dinethylformamide (DMF) can reach requirement during for solvent, but with pure N, dinethylformamide (DMF) is that the chloroform reference substance peak area of solvent is very little, and detection sensitivity is low.So determine the test sample disposal route be: sample thief is in tool plug test tube, and accurate the title decides, the accurate 75%N that adds, and dinethylformamide (DMF), close plug, jolting, centrifugal, precision is measured supernatant in 20ml head space bottle, rolls lid, promptly.

(5) linear relationship is investigated

Precision takes by weighing chloroform reference substance 0.1089g, adds 75%N, and dinethylformamide (DMF) dissolved dilution promptly gets the reference substance stock solution to 25ml.Accurate respectively 3ml, 5ml, 1ml, 3ml, 1ml, the 2ml of drawing, add 75%N, dinethylformamide (DMF) dilutes 2500 times, 2500 times, 250 times, 250 times, 25 times, 25 times respectively, get concentration and be respectively 0.0052272mg/ml, 0.008712mg/ml, 0.017424mg/ml, 0.052272mg/ml, 0.17424mg/ml, 0.34848mg/ml reference substance solution, precision is measured 2ml and is put into the head space bottle respectively again, rolls lid, sample introduction behind 110 ℃ of balance 30min in the head space stove, the record chromatogram.(mg/ml) is horizontal ordinate with reference substance solution, is that ordinate carries out linear regression with the peak area.Regression equation is: y=51031.5714x-0.8098, r=0.9999.The result shows that chloroform has good linear relationship in 5.2272 μ g～348.48 μ g scopes.See the following form:

Chloroform residual quantity linear relationship is investigated

With above two Equation for Calculating of same reference substance peak area (871.6) difference substitution, the RSD% of income value is 0.1%, therefore can calculate with one point external standard method.

(6) precision is investigated

Precision is measured reference substance solution (C=17.424 μ g/ml) 2ml and is put into the head space bottle respectively, rolls lid, sample introduction behind 110 ℃ of balance 30min in the head space stove, record chromatogram.6 parts of reference substance average peak area are 889.3, and RSD% is 2.2%, illustrate that precision is good.See the following form:

The precision test

(7) study on the stability

Measure chloroform reference substance solution (C=14.25 μ g/ml) 2ml respectively at 0,2,4,8,12 o'clock precisions and put into the head space bottle, roll lid, sample introduction behind 110 ℃ of balance 30min in the head space stove, record chromatogram.Recording the chloroform average peak area is 725.3, and RSD% is 5.4%, and according to the pharmacopeia regulation, this deviation is in specialized range.The result shows and has good stability.See the following form:

Study on the stability

(8) repeatability is investigated

The cantharides capsule 1.0g that gets the preparation method's preparation by compound cantharidin oral preparations of the present invention respectively is in 10ml tool plug test tube, the accurate 75%N that adds, dinethylformamide (DMF) 10ml, jolting 3min, centrifugal, precision is measured supernatant 2ml and is put into the head space bottle, rolls lid, sample introduction behind 110 ℃ of balance 30min in the head space stove, the record chromatogram.All do not detect chloroform in the results sample.The results are shown in following table:

Repeatability is investigated

(9) accuracy is investigated

Verify with the recovery.The cantharides capsule 1.0g that gets the preparation method's preparation by compound cantharidin oral preparations of the present invention respectively is in 10ml tool plug test tube, accurate chloroform reference substance solution (the C=17.424 μ g/ml) 10ml that adds, jolting 3min, centrifugal, precision is measured supernatant 2ml and is put into the head space bottle, add a cover sample introduction behind 110 ℃ of balance 30min in the head space stove, record chromatogram.Recording average average recovery is 98.3%, and RSD% is 3.0%, and the result shows that this method has good average recovery.The results are shown in following table: (A _Right=859.4)

Accuracy is investigated

(10) reappearance is investigated

Get 3 batches of samples respectively, by method of quality control mensuration of the present invention chloroform residual quantity wherein, there are two batches in three batch samples as a result and do not detect chloroform, the a collection of sample mean that detects chloroform is 0.001066%, RSD% is 6.43%, this deviation illustrates that this method reappearance is good in allowed band.See the following form: (C _{It is right to examine}=13.12 μ g/ml A _{It is right to examine}=962.2 C _Verify=14.91 μ g/ml A _Verify=1160.8)

Table 6 reappearance is investigated

(11) scope is investigated

The limit of chloroform residual quantity requires (must not be higher than 0.006%) in the preparation according to the present invention, and the scope of investigation is ± 50%.Get it filled respectively product 0.5g and 1.5g measure the chloroform residual quantity according to chloroform residual quantity inspection method in the method for quality control of the present invention, and the result does not extremely detect two of scope, and can reach described requirement.See the following form:

Scope is investigated

(12) sample determination

Precision takes by weighing sample 1.0g, puts in the 10ml tool plug test tube, the accurate 75%N that adds, and dinethylformamide (DMF) 10ml, jolting 3min, centrifugal, precision is measured supernatant 2ml in the head space bottle, rolls lid, puts into 110 ℃ of balance 30min of head space stove, sample introduction.Be calculated as follows chloroform residual quantity in the sample.

Measure chloroform residual quantity in 10 parts of compound cantharidin oral preparations by above method, the results are shown in following table: (chloroform reference substance concentration: C=14.91 μ g/ml, peak area: A _{On average}=1160.8)

The chloroform determination of residual amount

Above result has 5 parts not detect chloroform in 10 parts of compound cantharidin oral preparations as can be seen, has 5 parts to detect, and its content is also in the scope of national regulation.This method specificity is strong, highly sensitive, can effectively control the chloroform residual quantity in the preparation.

So will check in the method for quality control of the present invention that the residual limit of chloroform is decided to be under the item: the chloroform residual quantity must not cross 0.006%.

Compared with prior art, method of quality control of the present invention replenishes the assay project and the Sculellaria barbata of having set up cantharidin in the Radix Astragali and the Chinese blister beetle, the fruit of medicinal cornel, the thin layer of the fruit of glossy privet is differentiated project, increased the inspection of chloroform residual quantity, and the thin layer discrimination method of genseng and the Radix Astragali two flavor medicinal materials improved, thickness to developping agent and thin layer plate is selected, optimized discrimination method, its precision height, favorable reproducibility, good stability, recovery height, measurement result is accurate, improve the quality control standard of compound cantharidin oral system preparation, can control product quality effectively, thereby guaranteed its clinical efficacy.

Embodiment:

Further specify the present invention by the following examples, but not as limitation of the present invention.

Embodiments of the invention 1: the method for quality control of cantharides capsule agent comprises:

Proterties: its content is that yellow green is to tan powder, mildly bitter flavor Hui Tian.

Differentiate: (1) gets capsule content 3.5g, puts in the apparatus,Soxhlet's, adds an amount of chloroform, put in the water-bath refluxing extraction 2 hours, and discarded chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put in the apparatus,Soxhlet's again, add an amount of methyl alcohol, put in the water-bath refluxing extraction 4 hours, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 5ml makes dissolving, goes in the separating funnel, uses 2 washing containers of 5ml moisture again, be transferred in the separating funnel, extract 4 (10ml with water saturated normal butyl alcohol, 5ml, 5ml, 5ml), merge normal butyl alcohol liquid, wash with water 2 times, each 5ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 3 times with 0.8ml moisture, be transferred to neutral alumina post (100～200 orders, 2g, dry column-packing, diameter 1cm, long 25cm) on, with 30ml chloroform wash-out, discards the chloroform eluent earlier, use 70% methyl alcohol 25ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 1mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l put respectively on same silica gel g thin-layer plate (thick 500 μ m), and with methenyl choloride: methyl alcohol: lower floor's solution of water=13: 7: 2 is developping agent (controlled humidity in case of necessity), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 365nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

(2) get capsule content 2g, the 20ml that adds diethyl ether, reflux 30 minutes is put coldly, filters, and discards filtrate, and the dregs of a decoction add methyl alcohol 20ml, and reflux 30 minutes is put coldly, filters, and filtrate evaporate to dryness, residue add methyl alcohol 1ml dissolving, as need testing solution; Other gets sun plant control medicinal material 1g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 5 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl formate: formic acid=9: 5: 3 is developping agent, launch, take out, dry; Put under the 365nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

(3) get capsule content 3g, add 50% methyl alcohol 30ml, reflux 1 hour, put coldly, filter the filtrate evaporate to dryness, residue adds 50% methyl alcohol and dissolves in right amount, be added on the neutral alumina post (100～200 orders, 4g) on, with 40% methyl alcohol 50ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 10 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=4: 16: 2: 0.5 is developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

(4) get capsule content 3g, add ethyl acetate 20ml, ultrasonic 15min filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol 1ml dissolving, as need testing solution; Other gets fruit of glossy privet control medicinal material 0.5g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 5 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=20: 4: 0.5 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

Check: moisture must not cross 15.0%.

The chloroform residual quantity is shone gas chromatography determination:

Chromatographic condition and system suitability test fused-silica capillary column (column length 30m, internal diameter 0.25mm, film thickness 0.25 μ m) Rtx-5 (5% phenyl-methylsiloxane is a stationary phase); Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 110 ℃, heating 30min, 115 ℃ of sample introduction needle temperature, 120 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000.

The preparation of reference substance solution: precision takes by weighing the chloroform reference substance, adds 75%N, and dinethylformamide (DMF) dissolving is made the solution that every 1mL contains 15 μ g, promptly.

The preparation of need testing solution: get the about 1.0g of capsule content under the content uniformity item, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide (DMF) 10ml, close plug, jolting 3min, centrifugal, get supernatant, promptly.

Determination method is respectively accurate draws reference substance solution and each 2ml of need testing solution in 20ml head space bottle, rolls lid, behind 110 ℃ of balance 30min, gets saturated gas 1ml inject gas chromatograph mensuration in the head space bottle respectively, promptly in the head space stove.

In this capsule, the chloroform residual quantity must not cross 0.006%.

Other capsules of the present invention should meet Chinese Pharmacopoeia about the relevant regulations under the capsule item.

Assay: (1) Radix Astragali is according to high effective liquid chromatography for measuring: with the octadecylsilane chemically bonded silica is filling agent, and with acetonitrile: water=36: 64 is moving phase; Flow velocity 0.7ml/min; 40 ℃ of column temperatures; The evaporative light-scattering detector drift tube temperature is 90 ℃; Atomization gas is an air, and flow velocity is 2.2L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.2mg, promptly gets reference substance solution; Get the capsule under the content uniformity item, porphyrize is got about 1g, and accurate the title decides, put in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution 70ml, reflux 4 hours, extract evaporate to dryness, residue adds and is transferred in the separating funnel after water 20ml makes dissolving, with ethyl acetate extraction 2 times, and each 20ml; Combined ethyl acetate liquid adds water 15ml washing; Discard acetic acid ethyl fluid, merge water liquid, extract 4 times, each 40ml with water saturated normal butyl alcohol; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 40ml washing 1 time, normal butyl alcohol liquid evaporate to dryness, residue add water makes dissolving in right amount, is transferred to D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 12cm) on, water 50ml wash-out discards water liquid, use 70% ethanol 80ml wash-out again, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, filter with 0.45 μ m miillpore filter, promptly get need testing solution; The accurate respectively reference substance solution 5 μ l of absorption and 10 μ l, need testing solution 10 μ l inject hplc determination content; Contain the Radix Astragali in this capsule with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.12mg/g.

(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 (50% methyl-50% phenyl polysiloxane) immobile liquid is 1.5%, carrier Shimalite W (AW-DMCS) 80-100 order; Fid detector, injector temperature: 240 ℃; Detector temperature: 240 ℃; Column temperature is 155 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 40 μ g, promptly gets reference substance solution; Get the capsule content under the content uniformity item, porphyrize is got about 2g, the accurate title, decide, and puts in the 100ml beaker, adds the sodium chloride sodium hydroxide solution and (get 1mol/L sodium hydroxide solution 100mL and sodium chloride 2g, stir dissolving) 30ml, add 1ml acetone again, ultrasonic (power 250w, frequency 20kHz) 30 minutes put cold, add the 5ml concentrated hydrochloric acid, stir, leave standstill, put coldly, suction filtration washs residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction 3 times, each 30ml, combined chloroform liquid, put 50 ℃ of water-bath Back stroke to 1～2ml (chloroform must not be volatilized in the whole operation process), add chloroform again and be settled to 3ml, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 2 μ l of need testing solution respectively, and inject gas chromatograph is measured content; Contain cantharidin (C in this capsule ₁₀H ₁₂O ₄) be 0.028-0.160mg/g.

Embodiments of the invention 2: the method for quality control of cantharides tablet comprises:

Proterties: product is a Film coated tablets, removes to show yellow green behind the film-coating to sepia, mildly bitter flavor Hui Tian.

Differentiate: (1) gets tablet 3g, and porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put in the water-bath refluxing extraction 2 hours, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put in the water-bath refluxing extraction 3 hours, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 3ml makes dissolving, goes in the separating funnel, use 2 washing containers of 3ml moisture again, be transferred in the separating funnel, extract 3 times with water saturated normal butyl alcohol, each 10ml merges normal butyl alcohol liquid, washes with water 2 times, each 3ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 2 times with 0.6ml moisture, be transferred to neutral alumina post (100～200 orders, 2g, dry column-packing, diameter 1cm, long 25cm) on, with 20ml chloroform wash-out, discards the chloroform eluent earlier, use 70% methyl alcohol 20ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 0.8ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 0.8mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 15 μ l, reference substance solution 8 μ l put respectively on same silica gel g thin-layer plate (thick 450 μ m), and with methenyl choloride: methyl alcohol: lower floor's solution of water=20: 10: 5 is developping agent (controlled humidity in case of necessity), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 300nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

(2) get tablet 2.5g, porphyrize, the 25ml that adds diethyl ether, reflux 20 minutes is put coldly, filters, and discards filtrate, and the dregs of a decoction add methyl alcohol 25ml, and reflux 20 minutes is put coldly, filters, and filtrate evaporate to dryness, residue add methyl alcohol 0.8ml dissolving, as need testing solution; Other gets sun plant control medicinal material 0.8g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 10 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl formate: formic acid=10: 5: 5 is developping agent, launch, take out, dry; Put under the 300nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

(3) get tablet 2g, porphyrize adds 50% methyl alcohol 20ml, reflux 1.2 hours is put coldly, filters, filtrate evaporate to dryness, residue add 50% methyl alcohol and dissolve in right amount, are added on neutral alumina post (100～200 orders, 4g), with 40% methyl alcohol 35ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 0.8ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.8mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 12 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=5: 25: 5: 0.5 is developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

(4) get tablet 2g, porphyrize adds ethyl acetate 25ml, and ultrasonic 20min filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol 0.8ml dissolving, as need testing solution; Other gets fruit of glossy privet control medicinal material 1g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 10 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=30: 5: 0.5 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

Check: moisture must not cross 15.0%.

The chloroform residual quantity is shone gas chromatography determination: fused-silica capillary column (column length 30m, internal diameter 0.25mm, film thickness 0.25 μ m) Rtx-5 (5% phenyl methyl siloxane is a stationary phase); Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 105 ℃, heating 35min, 110 ℃ of sample introduction needle temperature, 115 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the solution that every 1mL contains 20 μ g is made in dinethylformamide (DMF) dissolving, promptly gets reference substance solution; Get the about 1.2g of tablet under the content uniformity item, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide (DMF) 12ml, close plug, jolting 2min, centrifugal, get supernatant, promptly get need testing solution; Respectively accurate reference substance solution and each 2.5ml of need testing solution of drawing rolls lid in 20ml head space bottle, behind 110 ℃ of balance 35min, get saturated gas 1.2ml inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this tablet, the chloroform residual quantity must not cross 0.006%.

Other tablets of the present invention should meet Chinese Pharmacopoeia about the relevant regulations under the tablet item.

Assay: (1) Radix Astragali is according to high effective liquid chromatography for measuring: with the octadecylsilane chemically bonded silica is filling agent, and with acetonitrile: water=50: 50 is moving phase; Flow velocity 0.6ml/min; 45 ℃ of column temperatures; The evaporative light-scattering detector drift tube temperature is 85 ℃; Atomization gas is an air, and flow velocity is 2.0L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.1mg, promptly gets reference substance solution; Get the tablet under the content uniformity item, porphyrize is got about 1.2g, and accurate the title decides, put in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution 75ml, reflux 5 hours, extract evaporate to dryness, residue adds and is transferred in the separating funnel after water 25ml makes dissolving, with ethyl acetate extraction 2 times, and each 25ml; Combined ethyl acetate liquid adds water 12ml washing; Discard acetic acid ethyl fluid, merge water liquid, extract 4 times, each 30ml with water saturated normal butyl alcohol; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 30ml washing 2 times, normal butyl alcohol liquid evaporate to dryness, residue add water makes dissolving in right amount, is transferred to D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 12cm) on, water 40ml wash-out discards water liquid, use 70% ethanol 70ml wash-out again, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, filter with 0.45 μ m miillpore filter, promptly get need testing solution; The accurate respectively reference substance solution 3 μ l of absorption and 8 μ l, need testing solution 8 μ l inject hplc determination content; Contain the Radix Astragali in this tablet with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.08mg/g.

(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 (50% methyl-50% phenyl polysiloxane) immobile liquid is 1.5%, carrier Shimalite W (AW-DMCS) 80-100 order; Fid detector, injector temperature: 235 ℃; Detector temperature: 235 ℃; Column temperature is 150 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 35 μ g, promptly gets reference substance solution; Get the tablet under the content uniformity item, porphyrize is got about 1.5g, the accurate title, decide, and puts in the 100ml beaker, adds sodium chloride-sodium hydroxide solution and (get 1mol/L sodium hydroxide solution 100mL and sodium chloride 2g, stir dissolving) 40ml, add 1.2ml acetone again, ultrasonic (power 250w, frequency 20kHz) 35 minutes put cold, add the 4ml concentrated hydrochloric acid, stir, leave standstill, put coldly, suction filtration washs residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction 3 times, each 35ml, combined chloroform liquid, put 50 ℃ of water-bath Back stroke to 1～2ml (chloroform must not be volatilized in the whole operation process), add chloroform again and be settled to 3ml, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 1 μ l of need testing solution respectively, and inject gas chromatograph is measured content; Contain cantharidin (C in this tablet ₁₀H ₁₂O ₄) be 0.025-0.180mg/g.

Embodiments of the invention 3: the method for quality control of cantharides granule comprises:

Proterties: product is that pale brown look is to auburn particle; It is sweet to distinguish the flavor of.

Differentiate: (1) gets granule 4g, and porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put in the water-bath refluxing extraction 2 hours, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put in the water-bath refluxing extraction 5 hours, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 7ml makes dissolving, goes in the separating funnel, use 2 washing containers of 7ml moisture again, be transferred in the separating funnel, extract 5 times with water saturated normal butyl alcohol, each 15ml merges normal butyl alcohol liquid, washes with water 2 times, each 7ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 4 times with 1.0ml moisture, be transferred to neutral alumina post (100～200 orders, 2g, dry column-packing, diameter 1cm, long 25cm) on, with 40ml chloroform wash-out, discards the chloroform eluent earlier, use 70% methyl alcohol 30ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 1.5ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 1.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 12 μ l, reference substance solution 6 μ l put respectively on same silica gel g thin-layer plate (thick 550 μ m), and with methenyl choloride: methyl alcohol: lower floor's solution of water=40: 15: 8 is developping agent (controlled humidity in case of necessity), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 400nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

(2) get granule 3g, porphyrize, the 30ml that adds diethyl ether, reflux 40 minutes is put coldly, filters, and discards filtrate, and the dregs of a decoction add methyl alcohol 30ml, and reflux 40 minutes is put coldly, filters, and filtrate evaporate to dryness, residue add methyl alcohol 1.5ml dissolving, as need testing solution; Other gets sun plant control medicinal material 1.5g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 15 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl formate: formic acid=15: 7: 4 is developping agent, launch, take out, dry; Put under the 400nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

(3) get granule 4g, porphyrize adds 50% methyl alcohol 40ml, reflux 1.5 hours is put coldly, filters, filtrate evaporate to dryness, residue add 50% methyl alcohol and dissolve in right amount, are added on neutral alumina post (100～200 orders, 4g), with 40% methyl alcohol 65ml wash-out, collect eluent, evaporate to dryness, residue adds methyl alcohol 1.5ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 1.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 15 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=7: 40: 8: 0.7 is developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

(4) get granule 4g, porphyrize adds ethyl acetate 30ml, and ultrasonic 25min filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol 1.5ml dissolving, as need testing solution; Other gets fruit of glossy privet control medicinal material 1.5g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 15 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=40: 7: 0.8 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

Check: moisture must not cross 15.0%.

The chloroform residual quantity is shone gas chromatography determination: fused-silica capillary column (column length 30m, internal diameter 0.25mm, film thickness 0.25 μ m) Rtx-5 (5% phenyl methyl siloxane is a stationary phase); Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cmm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 115 ℃, heating 30min, 120 ℃ of sample introduction needle temperature, 125 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the solution that every 1mL contains 25 μ g is made in dinethylformamide (DMF) dissolving, promptly gets reference substance solution; Get the about 1.5g of granule under the content uniformity item, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide (DMF) 15ml, close plug, jolting 4min, centrifugal, get supernatant, promptly get need testing solution; Respectively accurate reference substance solution and each 3ml of need testing solution of drawing rolls lid in 20ml head space bottle, behind 110 ℃ of balance 40min, get saturated gas 1.5ml inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this granule, the chloroform residual quantity must not cross 0.006%.

Other granules of the present invention should meet Chinese Pharmacopoeia about the relevant regulations under the granule item.

Assay: (1) Radix Astragali is according to high effective liquid chromatography for measuring: with the octadecylsilane chemically bonded silica is filling agent, and with acetonitrile: water=60: 40 is moving phase; Flow velocity 0.8ml/min; 50 ℃ of column temperatures; The evaporative light-scattering detector drift tube temperature is 95 ℃; Atomization gas is an air, and flow velocity is 2.5L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.3mg, promptly gets reference substance solution; Get the granule under the content uniformity item, porphyrize is got about 1.5g, and accurate the title decides, put in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution 80ml, reflux 5 hours, extract evaporate to dryness, residue adds and is transferred in the separating funnel after water 30ml makes dissolving, with ethyl acetate extraction 2 times, and each 30ml; Combined ethyl acetate liquid adds water 17ml washing; Discard acetic acid ethyl fluid, merge water liquid, extract 4 times, each 50ml with water saturated normal butyl alcohol; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 50ml washing 2 times, normal butyl alcohol liquid evaporate to dryness, residue add water makes dissolving in right amount, is transferred to D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 12cm) on, water 60ml wash-out discards water liquid, use 70% ethanol 90ml wash-out again, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, filter with 0.45 μ m miillpore filter, promptly get need testing solution; The accurate respectively reference substance solution 8 μ l of absorption and 15 μ l, need testing solution 15 μ l inject hplc determination content; Contain the Radix Astragali in this granule with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.01mg/g.

(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 (50% methyl-50% phenyl polysiloxane) immobile liquid is 1.5%, carrier Shimalite W (AW-DMCS) 80-100 order; Fid detector, injector temperature: 245 ℃; Detector temperature: 245 ℃; Column temperature is 160 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 45 μ g, promptly gets reference substance solution; Get the granule under the content uniformity item, porphyrize is got about 2.5g, the accurate title, decide, and puts in the 100ml beaker, adds sodium chloride-sodium hydroxide solution and (get 1mol/L sodium hydroxide solution 100mL and sodium chloride 2g, stir dissolving) 45ml, add 1.5ml acetone again, ultrasonic (power 250w, frequency 20kHz) 40 minutes put cold, add the 6ml concentrated hydrochloric acid, stir, leave standstill, put coldly, suction filtration washs residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction 3 times, each 35ml, combined chloroform liquid, put 50 ℃ of water-bath Back stroke to 1～2ml (chloroform must not be volatilized in the whole operation process), add chloroform again and be settled to 3ml, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 3 μ l of need testing solution respectively, and inject gas chromatograph is measured content; Contain cantharidin (C in this granule ₁₀H ₁₂O ₄) be 0.003-0.018mg/g.

Embodiments of the invention 4: the method for quality control of compound mylabris preparation can comprise:

For granule, product is that pale brown look is to auburn particle; It is sweet to distinguish the flavor of.

Differentiate: (1) gets capsule, each 2g of tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put in the water-bath refluxing extraction 1 hour, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put in the water-bath refluxing extraction 2 hours, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 1ml makes dissolving, goes in the separating funnel, uses 1ml water washing container again, be transferred in the separating funnel, extract 2 times with water saturated normal butyl alcohol, each 2ml merges normal butyl alcohol liquid, wash with water 1 time, each 1ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 1 time with 0.4ml water, be transferred to 2g, dry column-packing, diameter 1cm, long 25cm, on 100～200 purpose neutral alumina posts, with 10ml chloroform wash-out, discard the chloroform eluent earlier, use 70% methyl alcohol 10ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 0.5ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 0.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 5-20 μ l, reference substance solution 2-10 μ l puts respectively on same silica gel g thin-layer plate (thick 450 μ m), and with methenyl choloride: methyl alcohol: lower floor's solution of water=5: 20: 0.5 is developping agent (controlled humidity in case of necessity), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 200nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

(2) get capsule, tablet or granule 1g, porphyrize, the 10ml that adds diethyl ether, reflux 10 minutes is put cold, filter, discard filtrate, the dregs of a decoction add methyl alcohol 10ml, and reflux 10 minutes is put cold, filter, filtrate evaporate to dryness, residue add methyl alcohol 0.5ml dissolving, as need testing solution; Other gets sun plant control medicinal material 0.5g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 2 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl formate: formic acid=2: 9: 1 is developping agent, launch, take out, dry; Put under the 200nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

Check: moisture must not cross 15.0%.

Other capsules of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the relevant regulations under capsule, tablet or the granule item.

Assay: cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 (50% methyl, 50% phenyl polysiloxane) immobile liquid is 1.5%, carrier Shimalite W (AW-DMCS) 80 100 orders; Fid detector, injector temperature: 230 ℃; Detector temperature: 230 ℃; Column temperature is 150 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 30 μ g, promptly gets reference substance solution; Get the capsule under the content uniformity item, tablet or granule content, porphyrize is got about 1g, the accurate title, decide, and puts in the 100ml beaker, adds sodium chloride-sodium hydroxide solution and (get 1mol/L sodium hydroxide solution 100mL and sodium chloride 2g, stir dissolving) 30ml, add 0.5ml acetone again, ultrasonic (power 250w, frequency 20kHz) 20 minutes put cold, add the 2ml concentrated hydrochloric acid, stir, leave standstill, put coldly, suction filtration washs residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction 2 times, each 30ml, combined chloroform liquid, put 50 ℃ of water-bath Back stroke to 1～2ml (chloroform must not be volatilized in the whole operation process), add chloroform again and be settled to 3ml, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 0.5 μ l of need testing solution respectively, and inject gas chromatograph is measured content; In this oral formulations, contain cantharidin (C in the capsule ₁₀H ₁₂O ₄) be 0.025-0.180mg/g; Contain cantharidin (C in the tablet ₁₀H ₁₂O ₄) be 0.025-0.180mg/g; Contain cantharidin (C in the granule ₁₀H ₁₂O ₄) be 0.003-0.018mg/g.

Embodiments of the invention 5: the method for quality control of compound mylabris preparation can comprise:

Differentiate: (1) gets capsule, tablet or granule 5g, and porphyrize adds 50% methyl alcohol 10ml, reflux 0.5 hour is put coldly, filters, the filtrate evaporate to dryness, residue adds 50% methyl alcohol and dissolves in right amount, is added on the neutral alumina post, with 40% methyl alcohol 20ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 0.5ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 5 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=1: 50: 0.5: 0.9 is developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

(2) get capsule, tablet or granule 1g, porphyrize adds ethyl acetate 10ml, and ultrasonic 10min filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol 0.5ml dissolving, as need testing solution; Other gets fruit of glossy privet control medicinal material 0.2g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 2 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10: 9: 0.1 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

Check: moisture must not cross 15.0%.

Assay: the Radix Astragali is according to high effective liquid chromatography for measuring: chromatographic column is the C4 post, and with acetonitrile: water=10: 90 is moving phase; Flow velocity 0.5ml/min; 30 ℃ of column temperatures; The evaporative light-scattering detector drift tube temperature is 80 ℃; Atomization gas is an air, and flow velocity is 1.5L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.05mg, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, porphyrize, get about 0.5g, the accurate title, decide, and puts in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution 50ml, reflux 4 hours, extract evaporate to dryness, residue add and are transferred in the separating funnel after water 10ml makes dissolving, extract 1 time with ethyl acetate 40ml, acetic acid ethyl fluid adds water 10ml washing; Discard acetic acid ethyl fluid, merge water liquid, extract 3 times, each 20ml with water saturated normal butyl alcohol; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 20ml washing 1 time, normal butyl alcohol liquid evaporate to dryness, residue add water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins, water 30ml wash-out discards water liquid, uses 70% ethanol 60ml wash-out again, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, filter, promptly get need testing solution with 0.45 μ m miillpore filter; The accurate respectively reference substance solution 1 μ l of absorption and 2 μ l, need testing solution 2 μ l inject hplc determination content; In this oral formulations, contain the Radix Astragali in the capsule with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.01mg/g.

Embodiments of the invention 6: the method for quality control of compound mylabris preparation can comprise:

Differentiate: get capsule, tablet or granule 5g, porphyrize adds ethyl acetate 40ml, and ultrasonic 30min filters, and filtrate evaporate to dryness, residue add absolute ethyl alcohol 2ml dissolving, as need testing solution; Other gets fruit of glossy privet control medicinal material 2g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 20 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=50: 1: 0.9 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 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

Check: moisture must not cross 15.0%.

The chloroform residual quantity is shone gas chromatography determination: fused-silica capillary column (column length 30m, internal diameter 0.25mm, film thickness 0.25 μ m) Rtx-5 (5% phenyl methyl siloxane is a stationary phase); Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100 ℃, heating 25min, 110 ℃ of sample introduction needle temperature, 110 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the solution that every 1mL contains 10 μ g is made in dinethylformamide (DMF) dissolving, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, get the about 0.5g of content, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide (DMF) 5ml, close plug, jolting 1min, centrifugal, get supernatant, promptly get need testing solution; Respectively accurate reference substance solution and each 1ml of need testing solution of drawing rolls lid in 20ml head space bottle, behind 110 ℃ of balance 20min, get saturated gas 0.5ml inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%.

Assay: (1) Radix Astragali is according to high effective liquid chromatography for measuring: chromatographic column is the C8 post, and with acetonitrile: water=90: 10 is moving phase; Flow velocity 1.0ml/min; 60 ℃ of column temperatures; The evaporative light-scattering detector drift tube temperature is 100 ℃; Atomization gas is an air, and flow velocity is 3.0L/min; Number of theoretical plate calculates by the Astragaloside IV peak should be not less than 3000; Precision takes by weighing the Astragaloside IV reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5mg, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, porphyrize, get about 2g, the accurate title, decide, and puts in the apparatus,Soxhlet's, add 2% potassium hydroxide methanol solution 100ml, reflux 6 hours, extract evaporate to dryness, residue add and are transferred in the separating funnel after water 40ml makes dissolving, with ethyl acetate extraction 3 times, each 10ml; Combined ethyl acetate liquid adds water 20ml washing; Discard acetic acid ethyl fluid, merge water liquid, extract 5 times, each 60ml with water saturated normal butyl alcohol; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 60ml washing 2 times, normal butyl alcohol liquid evaporate to dryness, residue add water makes dissolving in right amount, is transferred to D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 12cm) on, water 70ml wash-out discards water liquid, use 70% ethanol 100ml wash-out again, collect eluent, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methyl alcohol to scale, shake up, filter with 0.45 μ m miillpore filter, promptly get need testing solution; The accurate respectively reference substance solution 10 μ l of absorption and 20 μ l, need testing solution 20 μ l inject hplc determination content; In this oral formulations, contain the Radix Astragali in the capsule with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with Astragaloside IV (C ₄₁H ₆₈O ₁₄) meter, must not be less than 0.01mg/g.

(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 (50% methyl-50% phenyl polysiloxane) immobile liquid is 1.5%, carrier Shimalite W (AW-DMCS) 80-100 order; Fid detector, injector temperature: 250 ℃; Detector temperature: 250 ℃; Column temperature is 160 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 50 μ g, promptly gets reference substance solution; Get the capsule under the content uniformity item, tablet or granule content, porphyrize is got about 3g, the accurate title, decide, and puts in the 100ml beaker, adds the sodium chloride sodium hydroxide solution and (get 1mol/L sodium hydroxide solution 100mL and sodium chloride 2g, stir dissolving) 50ml, add 2ml acetone again, ultrasonic (power 250w, frequency 20kHz) 50 minutes put cold, add the 8ml concentrated hydrochloric acid, stir, leave standstill, put coldly, suction filtration washs residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction 4 times, each 40ml, combined chloroform liquid, put 50 ℃ of water-bath Back stroke to 1～2ml (chloroform must not be volatilized in the whole operation process), add chloroform again and be settled to 3ml, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 5 μ l of need testing solution respectively, and inject gas chromatograph is measured content; In this oral formulations, contain cantharidin (C in the capsule ₁₀H ₁₂O ₄) be 0.025-0.180mg/g; Contain cantharidin (C in the tablet ₁₀H ₁₂O ₄) be 0.025-0.180mg/g; Contain cantharidin (C in the granule ₁₀H ₁₂O ₄) be 0.003-0.018mg/g.

Embodiments of the invention 7: the method for quality control of compound mylabris preparation can comprise:

Differentiate: (1) gets capsule, each 5g of tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put in the water-bath refluxing extraction 3 hours, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put in the water-bath refluxing extraction 6 hours, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 10ml makes dissolving, goes in the separating funnel, uses 3 washing containers of 10ml moisture again, be transferred in the separating funnel, extract 6 times with water saturated normal butyl alcohol, each 20ml merges normal butyl alcohol liquid, wash with water 3 times, each 10ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 5 times with 1.2ml moisture, be transferred to 2g, dry column-packing, diameter 1cm, long 25cm, on 100～200 purpose neutral alumina posts, with 50ml chloroform wash-out, discard the chloroform eluent earlier, use 70% methyl alcohol 40ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 20 μ l, reference substance solution 10 μ l put respectively on same silica gel g thin-layer plate (thick 550 μ m), and with methenyl choloride: methyl alcohol: lower floor's solution of water=50: 2: 10 is developping agent (controlled humidity in case of necessity), launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.

(2) get capsule, tablet or granule 4g, porphyrize, the 40ml that adds diethyl ether, reflux 50 minutes is put cold, filter, discard filtrate, the dregs of a decoction add methyl alcohol 40ml, and reflux 50 minutes is put cold, filter, filtrate evaporate to dryness, residue add methyl alcohol 2ml dissolving, as need testing solution; Other gets sun plant control medicinal material 2g, shines medicinal material solution in pairs with legal system; According to Chinese Pharmacopoeia thin-layered chromatography test, draw need testing solution, each 20 μ l of control medicinal material solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: ethyl formate: formic acid=20: 1: 9 is developping agent, launch, take out, dry; Put under the 500nm ultraviolet lamp and inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.

(3) get capsule, tablet or granule 5g, porphyrize adds 50% methyl alcohol 50ml, reflux 2 hours is put coldly, filters, the filtrate evaporate to dryness, residue adds 50% methyl alcohol and dissolves in right amount, is added on the neutral alumina post, with 40% methyl alcohol 80ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 20 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=9: 5: 10: 0.1 is developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.

Check: moisture must not cross 15.0%.

The chloroform residual quantity is shone gas chromatography determination: fused-silica capillary column (column length 30m, internal diameter 0.25mm, film thickness 0.25 μ m) Rtx-5 (5% phenyl methyl siloxane is a stationary phase); Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 120 ℃, heating 40min, 120 ℃ of sample introduction needle temperature, 130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the solution that every 1mL contains 30 μ g is made in dinethylformamide (DMF) dissolving, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, get the about 2.0g of content, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide (DMF) 20ml, close plug, jolting 5min, centrifugal, get supernatant, promptly get need testing solution; Respectively accurate reference substance solution and each 4ml of need testing solution of drawing rolls lid in 20ml head space bottle, behind 110 ℃ of balance 50min, get saturated gas 2.0ml inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%.

Claims

1. the detection method of a compound cantharidin oral preparations, described oral formulations is capsule, tablet or granule, it is characterized in that: described detection method is proterties, discriminating, inspection and assay project; Wherein in the proterties project, for capsule, content is that yellow green is to tan powder, mildly bitter flavor Hui Tian, for tablet, product is a Film coated tablets, removes to show yellow green behind the film-coating to sepia, mildly bitter flavor Hui Tian, for granule, product is that pale brown look is to auburn particle; It is sweet to distinguish the flavor of; In the discriminating project, the discrimination method of the genseng and the Radix Astragali is to be contrast with ginsenoside Rg1's reference substance and Astragaloside IV reference substance, with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is the thin-layered chromatography of developping agent, the discrimination method of Sculellaria barbata is to be contrast with the Sculellaria barbata control medicinal material, with toluene: ethyl formate: formic acid=2-20: 1-9: 1-9 is the thin-layered chromatography of developping agent, the discrimination method of the fruit of medicinal cornel is to be contrast with the loganin reference substance, with toluene: acetone: absolute ethyl alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is the thin-layered chromatography of developping agent, the discrimination method of the fruit of glossy privet is to be contrast with fruit of glossy privet control medicinal material, and with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is the thin-layered chromatography of developping agent; Inspection item is moisture, chloroform residual quantity and other conventional projects, and wherein the inspection method of chloroform residual quantity is to be the vapor-phase chromatography of contrast with the chloroform reference substance; In the assay project, the content assaying method of the Radix Astragali is to be contrast with the Astragaloside IV reference substance, with acetonitrile: water=10-90: 90-10 is the high performance liquid chromatography of moving phase, and the content assaying method of contained cantharidin is to be the vapor-phase chromatography of contrast with the cantharidin reference substance in the Chinese blister beetle.

2. according to the detection method of the described compound cantharidin oral preparations of claim 1, it is characterized in that: described discrimination method is:

(1) gets capsule, tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put refluxing extraction in the water-bath, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put refluxing extraction in the water-bath, reclaim methyl alcohol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separating funnel, wash container again with water, be transferred in the separating funnel, extract with water saturated normal butyl alcohol, extract washes with water, divide and get normal butyl alcohol liquid, evaporate to dryness, the dissolving of residue water is transferred on the neutral alumina post, use earlier the chloroform wash-out, discard the chloroform eluent, use methanol-eluted fractions again, eluent is evaporate to dryness in water-bath, residue adds methyl alcohol makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is developping agent, launches, and takes 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 ℃, puts under the 200-500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(3) get capsule, tablet or granule, porphyrize adds methyl alcohol, and reflux is put cold, filter, filtrate evaporate to dryness, residue add methyl alcohol and dissolve in right amount, are added on the neutral alumina post, use methanol-eluted fractions, collect eluent, evaporate to dryness, residue add methyl alcohol makes dissolving, as need testing solution; Other gets the loganin reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is a developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;

3. according to the detection method of the described compound cantharidin oral preparations of claim 2, it is characterized in that: discrimination method is more specifically:

(1) gets capsule, each 2-5g of tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put in the water-bath refluxing extraction 1-3 hour, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put in the water-bath refluxing extraction 2-6 hour, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 1-10ml makes dissolving, goes in the separating funnel, uses 1-3 washing container of 1-10ml moisture again, be transferred in the separating funnel, extract 2-6 time with water saturated normal butyl alcohol, each 2-20ml merges normal butyl alcohol liquid, wash with water 1-3 time, each 1-10ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 1-5 time with 0.4-1.2ml moisture, be transferred to 2g, dry column-packing, diameter 1cm, long 25cm, on 100～200 purpose neutral alumina posts, with 10-50ml chloroform wash-out, discard the chloroform eluent earlier, use 70% methyl alcohol 10-40ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 0.5-2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 5-20 μ l, reference substance solution 2-10 μ l puts respectively on the thick same silica gel g thin-layer plate of 450-550 μ m, and with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 200-500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

(3) get capsule, tablet or granule 1-5g, porphyrize adds 50% methyl alcohol 10-50ml, reflux 0.5-2 hour, put coldly, filter, the filtrate evaporate to dryness, residue adds 50% methyl alcohol and dissolves in right amount, is added on 4g, 100～200 purpose neutral alumina posts, with 40% methyl alcohol 20-80ml wash-out, collect eluent, evaporate to dryness, residue add methyl alcohol 0.5-2ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methyl alcohol and makes the solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution, each 5-20 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose, with toluene: acetone: absolute ethyl alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is a developping agent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;

4. according to the detection method of the described compound cantharidin oral preparations of claim 1, it is characterized in that: the inspection method of chloroform residual quantity is: shine gas chromatography determination: the fused-silica capillary column Rtx-5 of column length 30m, internal diameter 0.25mm, thickness 0.25 μ m is a stationary phase with 5% phenyl-methylsiloxane; Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100-120 ℃, heating 25-40min, 110-120 ℃ of sample introduction needle temperature, 110-130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the dinethylformamide dissolving promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide, close plug, jolting, centrifugal, get supernatant, promptly get need testing solution; Accurate respectively absorption reference substance solution and need testing solution roll lid in the head space bottle, after high temperature balance a period of time, get saturated gas inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%.

5. according to the detection method of the described compound cantharidin oral preparations of claim 4, it is characterized in that: the inspection method of chloroform residual quantity is more specifically: shine gas chromatography determination: the fused-silica capillary column Rtx-5 of column length 30m, internal diameter 0.25mm, thickness 0.25 μ m is a stationary phase with 5% phenyl-methylsiloxane; Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100-120 ℃, heating 25-40min, 110-120 ℃ of sample introduction needle temperature, 110-130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the solution that every 1mL contains 10-30 μ g is made in the dinethylformamide dissolving, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, get the about 0.5-2.0g of content, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide 5-20ml, close plug, jolting 1-5min, centrifugal, get supernatant, promptly get need testing solution; Respectively accurate reference substance solution and each 1-4ml of need testing solution of drawing rolls lid in 20ml head space bottle, behind 110 ℃ of balance 20-50min, get saturated gas 0.5-2.0ml inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%.

6. according to the detection method of the described compound cantharidin oral preparations of claim 1, it is characterized in that: described content assaying method is:

(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 immobile liquid is 1.5%, carrier Shimalite W 80-100 order; Fid detector, injector temperature: 230-250 ℃; Detector temperature: 230-250 ℃; Column temperature is 150-160 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and adds the chloroform dissolving, shakes up, and promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, the accurate title, decide, and puts in the beaker, adds sodium chloride-sodium hydroxide solution, add acetone again, ultrasonic, put cold, add concentrated hydrochloric acid, stir, leave standstill, put coldly, suction filtration washs residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction, and chloroform solution is put 50 ℃ of water-bath Back stroke to a small amount of, add the chloroform constant volume again, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 0.5-5 μ l of need testing solution respectively, and inject gas chromatograph is measured content; In this oral formulations, contain cantharidin C in the capsule ₁₀H ₁₂O ₄Be 0.025-0.180mg/g; Contain cantharidin C in the tablet ₁₀H ₁₂O ₄Be 0.025-0.180mg/g; Contain cantharidin C in the granule ₁₀H ₁₂O ₄Be 0.003-0.018mg/g.

7. according to the detection method of the described compound cantharidin oral preparations of claim 6, it is characterized in that: content assaying method is more specifically:

(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm i d, and the stain amount that is coated with of OV-17 50% methyl-50% phenyl polysiloxane immobile liquid is 1.5%, carrier Shimalite W 80-100 order; Fid detector, injector temperature: 230-250 ℃; Detector temperature: 230-250 ℃; Column temperature is 150-160 ℃; Number of theoretical plate calculates by the cantharidin peak should be not less than 1000; Get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 30-50 μ g, promptly gets reference substance solution; Get capsule, tablet or granule content under the content uniformity item, porphyrize is got about 1-3g, the accurate title, decide, and puts in the 100ml beaker, adds sodium chloride-sodium hydroxide solution 30-50ml, add 0.5-2ml acetone again, under power 250w, the frequency 20kHz ultrasonic 20-50 minute, put cold, add the 2-8ml concentrated hydrochloric acid, stir, leave standstill, put cold, suction filtration, wash residue with low amounts of water, merging filtrate, filtrate is used chloroform extraction 2-4 time, each 30-40ml, combined chloroform liquid is put 50 ℃ of water-bath Back stroke to 1～2ml, adds chloroform again and is settled to 3ml, shake up, promptly get need testing solution; Precision is measured reference substance solution and each 0.5-5 μ l of need testing solution respectively, and inject gas chromatograph is measured content; In this oral formulations, contain cantharidin C in the capsule ₁₀H ₁₂O ₄Be 0.025-0.180mg/g; Contain cantharidin C in the tablet ₁₀H ₁₂O ₄Be 0.025-0.180mg/g; Contain cantharidin C in the granule ₁₀H ₁₂O ₄Be 0.003-0.018mg/g.

8. according to the detection method of the described compound cantharidin oral preparations of claim 1, it is characterized in that: described detection method is:

Differentiate: (1) gets capsule, tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put refluxing extraction in the water-bath, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put refluxing extraction in the water-bath, reclaim methyl alcohol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separating funnel, wash container again with water, be transferred in the separating funnel, extract with water saturated normal butyl alcohol, extract washes with water, divide and get normal butyl alcohol liquid, evaporate to dryness, the dissolving of residue water is transferred on the neutral alumina post, use earlier the chloroform wash-out, discard the chloroform eluent, use methanol-eluted fractions again, eluent is evaporate to dryness in water-bath, residue adds methyl alcohol makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is developping agent, launches, and takes 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 ℃, puts under the 200-500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Check: moisture must not cross 15.0%;

The chloroform residual quantity is shone gas chromatography determination: the fused-silica capillary column Rtx-5 of column length 30m, internal diameter 0.25mm, thickness 0.25 μ m is a stationary phase with 5% phenyl-methylsiloxane; Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100-120 ℃, heating 25-40min, 110-120 ℃ of sample introduction needle temperature, 110-130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the dinethylformamide dissolving promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide, close plug, jolting, centrifugal, get supernatant, promptly get need testing solution; Accurate respectively absorption reference substance solution and need testing solution roll lid in the head space bottle, after high temperature balance a period of time, get saturated gas inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%;

9. according to the detection method of the described compound cantharidin oral preparations of claim 8, it is characterized in that: described detection method is:

Differentiate: (1) gets capsule, each 2-5g of tablet or granule, porphyrize is put in the apparatus,Soxhlet's, add an amount of chloroform, put in the water-bath refluxing extraction 1-3 hour, discard chloroform solution, take out filter cylinder, dry, treat that chloroform waves to the greatest extent, put again in the apparatus,Soxhlet's, add an amount of methyl alcohol, put in the water-bath refluxing extraction 2-6 hour, reclaim methyl alcohol to doing, residue adds 5% sodium hydroxide solution 1-10ml makes dissolving, goes in the separating funnel, uses 1-3 washing container of 1-10ml moisture again, be transferred in the separating funnel, extract 2-6 time with water saturated normal butyl alcohol, each 2-20ml merges normal butyl alcohol liquid, wash with water 1-3 time, each 1-10ml divides and gets normal butyl alcohol liquid, evaporate to dryness, residue dissolves for 1-5 time with 0.4-1.2ml moisture, be transferred to 2g, dry column-packing, diameter 1cm, long 25cm, on 100～200 purpose neutral alumina posts, with 10-50ml chloroform wash-out, discard the chloroform eluent earlier, use 70% methyl alcohol 10-40ml wash-out again, eluent is evaporate to dryness in water-bath, and residue adds methyl alcohol 0.5-2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and Astragaloside IV reference substance, adds methyl alcohol and makes the mixed solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin-layered chromatography, draw need testing solution 5-20 μ l, reference substance solution 2-10 μ l puts respectively on the thick same silica gel g thin-layer plate of 450-550 μ m, and with methenyl choloride: lower floor's solution of methyl alcohol: water=5-50: 2-20: 0.5-10 is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot colour developing at 105 ℃, puts under the 200-500nm ultraviolet lamp and inspects; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color;

Check: moisture must not cross 15.0%;

The chloroform residual quantity is shone gas chromatography determination: the fused-silica capillary column Rtx-5 of column length 30m, internal diameter 0.25mm, thickness 0.25 μ m is a stationary phase with 5% phenyl-methylsiloxane; Temperature programme: 60 ℃ of initial temperatures, keep 5min, be warming up to 180 ℃ with the speed of 30 ℃ of per minutes, keep 5min; Fid detector; Injector temperature: 200 ℃; Detector temperature: 250 ℃; Split ratio: 10: 1; Linear velocity: 30cm/sec; Hydrogen flowing quantity: 40mL/min; Air mass flow: 400mL/min; Adopt headspace sampling: the vaporizer temperature is 100-120 ℃, heating 25-40min, 110-120 ℃ of sample introduction needle temperature, 110-130 ℃ of transfer tube temperature; Number of theoretical plate calculates with the chloroform peak should be not less than 5000; Precision takes by weighing the chloroform reference substance, adds 75%N, and the solution that every 1mL contains 10-30 μ g is made in the dinethylformamide dissolving, promptly gets reference substance solution; Get capsule, tablet or granule under the content uniformity item, get the about 0.5-2.0g of content, the accurate title, decide, and puts in the tool plug test tube, the accurate 75%N that adds, and dinethylformamide 5-20ml, close plug, jolting 1-5min, centrifugal, get supernatant, promptly get need testing solution; Respectively accurate reference substance solution and each 1-4ml of need testing solution of drawing rolls lid in 20ml head space bottle, behind 110 ℃ of balance 20-50min, get saturated gas 0.5-2.0ml inject gas chromatograph mensuration in the head space bottle respectively in the head space stove; In this oral formulations, the chloroform residual quantity must not cross 0.006%;

10. according to the detection method of claim 7,8 or 9 described compound cantharidin oral preparations, it is characterized in that: used sodium chloride-sodium hydroxide solution is to add sodium chloride 2g in 1mol/L sodium hydroxide solution 100mL, and stirring, dissolving form.