CN101036774A - Quality control method of compound cantharidin oral preparations - Google Patents
Quality control method of compound cantharidin oral preparations Download PDFInfo
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- CN101036774A CN101036774A CNA2007102004651A CN200710200465A CN101036774A CN 101036774 A CN101036774 A CN 101036774A CN A2007102004651 A CNA2007102004651 A CN A2007102004651A CN 200710200465 A CN200710200465 A CN 200710200465A CN 101036774 A CN101036774 A CN 101036774A
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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
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 tumor 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 tumor.In research process, effectively filter out medicine and just seem particularly important with antitumor action.Compound cantharidin oral preparations by Mylabris, Radix Ginseng, the Radix Astragali, Radix Et Caulis Acanthopanacis Senticosi, rhizoma sparganic, Herba Scutellariae Barbatae, Rhizoma Curcumae, Fructus Corni, Fructus Ligustri Lucidi, Fel Ursi powder, Radix Glycyrrhizae totally ten simply medicine be prepared from, has the removing blood stasis repercussive, the effect of counteracting toxic substances phagedenoma is mainly used in diseases such as primary hepatocarcinoma, pulmonary carcinoma, rectal cancer, malignant lymphoma, gynecologic malignant tumor.Wherein " FUFANG BANMAO JIAONANG " is on the books in the 17 in health ministry drug standard 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.Herba Scutellariae Barbatae, Fructus Corni, Fructus Ligustri Lucidi etc. are effective ingredient 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 Radix Ginseng and the Radix Astragali two flavor medical materials differentiated, the selection of developing solvent was not ideal enough, causes standing time longer, and operation inconvenience, and because factor affecting such as operating environments causes in discrimination process separating degree bad, and the speckle 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 ingredient 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 Herba Scutellariae Barbatae, Fructus Corni, Fructus Ligustri Lucidi is differentiated project, increased the inspection of chloroform residual quantity, and the thin layer discrimination method of Radix Ginseng and the Radix Astragali two flavor medical materials improved, thickness to developing solvent and lamellae 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 Mylabris 4-12, Radix Ginseng 10-30, Radix Astragali 50-150, Radix Et Caulis Acanthopanacis Senticosi 50-150, rhizoma sparganic 16-48, Herba Scutellariae Barbatae 60-180, Rhizoma Curcumae 16-48, Fructus Corni 20-60, Fructus Ligustri Lucidi 20-60, Fel Ursi powder 0.4-1.2 and Radix Glycyrrhizae 10-30.Its preparation method is: above ten simply, and except that Fel Ursi powder, Radix Ginseng, Fructus Corni, Fructus Ligustri Lucidi, Herba Scutellariae Barbatae are ground into fine powder, sieve, and be standby; Mylabris is soaked with chloroform and extracts 1-5 time, and each 16-48ml soaked 48-96 hour, and merge extractive liquid, reclaims chloroform, is concentrated into the thick paste shape; The five tastes such as all the other Radixs Astragali decoct with water 1-5 time, and each 0.5-5 hour, collecting decoction filtered, and filtrate is concentrated into relative density and is about 1.00-1.40 (60-100 ℃); Fel Ursi powder adds fine powders such as above-mentioned thick paste, concentrated solution and Radix Ginseng after adding 80 ℃ of water dissolutioies, stirs evenly, and in oven dry below 100 ℃, is ground into fine powder, adds adjuvant, makes capsule, tablet or granule with conventional method.Adjuvant can be added in this preparation, also adjuvant can be do not added.
Method of quality control of the present invention mainly comprise in character, discriminating, inspection and the assay project partly or entirely; Wherein differentiate the thin layer chromatography discriminating that comprises Radix Ginseng, the Radix Astragali, Herba Scutellariae Barbatae, Fructus Corni and Fructus Ligustri Lucidi 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 Mylabris.
The discrimination method of the Radix Ginseng and the Radix Astragali is to be contrast with ginsenoside Rg1's reference substance and astragaloside reference substance, and with chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is the thin layer chromatography of developing solvent; The discrimination method of Herba Scutellariae Barbatae is to be contrast with the Herba Scutellariae Barbatae control medicinal material, and with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is the thin layer chromatography of developing solvent; The discrimination method of Fructus Corni is to be contrast with the loganin reference substance, and with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is the thin layer chromatography of developing solvent; The discrimination method of Fructus Ligustri Lucidi is to be contrast with the Fructus Ligustri Lucidi control medicinal material, and with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is the thin layer chromatography of developing solvent; The inspection method of chloroform residual quantity is to be the gas chromatography of contrast with the chloroform reference substance; The content assaying method of the Radix Astragali is to be contrast with the astragaloside reference substance, is the high performance liquid chromatography of mobile phase with acetonitrile: water=10-90: 90-10; The content assaying method of contained cantharidin is to be the gas chromatography of contrast with the cantharidin reference substance in the Mylabris.
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 reflux, extract, 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 methanol, put reflux, extract, in the water-bath, reclaim methanol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separatory funnel, reuse water washing container, be transferred in the separatory funnel, use water saturated n-butanol extraction, extracting solution washes with water, divide and get n-butyl alcohol liquid, evaporate to dryness, the residue water dissolution is transferred on the neutral alumina post, use earlier the chloroform eluting, discard the chloroform eluent, the reuse methanol-eluted fractions, eluent is evaporate to dryness in water-bath, residue adds methanol makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer chromatography, draw need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 200-500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
(2) get capsule, tablet or granule, porphyrize, the reflux that adds diethyl ether is put coldly, filters, and discards filtrate, and medicinal residues add the methanol reflux, put coldly, filter, the filtrate evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Other gets the Herba Portulacae Grandiflorae control medicinal material, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is developing solvent, launch, take out, dry; Put under the 200-500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(3) get capsule, tablet or granule, porphyrize adds methanol, and reflux is put cold, filter, filtrate evaporate to dryness, residue add methanol and dissolve in right amount, are added on the neutral alumina post, use methanol-eluted fractions, collect eluent, evaporate to dryness, residue add methanol 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 layer chromatography, draw need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 the Fructus Ligustri Lucidi control medicinal material, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 2-6 hour, reclaim methanol to doing, residue adds 5% sodium hydroxide solution 1-10ml makes dissolving, goes in the separatory funnel 1-3 washing container of reuse 1-10ml moisture, be transferred in the separatory funnel, with water saturated n-butanol extraction 2-6 time, each 2-20ml merges n-butyl alcohol liquid, wash with water 1-3 time, each 1-10ml divides and gets n-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 eluting, discard the chloroform eluent earlier, reuse 70% methanol 10-40ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 0.5-2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 200-500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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, medicinal residues add methanol 10-40ml, reflux 10-50 minute, put cold, filter, filtrate evaporate to dryness, residue add methanol 0.5-2ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 0.5-2g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is developing solvent, launch, take out, dry; Put under the 200-500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(3) get capsule, tablet or granule 1-5g, porphyrize adds 50% methanol 10-50ml, reflux 0.5-2 hour, put coldly, filter, the filtrate evaporate to dryness, residue adds 50% methanol and dissolves in right amount, is added on 4g, 100～200 purpose neutral alumina posts, with 40% methanol 20-80ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5-2ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle 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 dehydrated alcohol 0.5-2ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 0.2-2g, shines medical material solution in pairs with legal system; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 an immobile phase with 5% phenyl-methylsiloxane; Temperature programming: 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 an immobile phase with 5% phenyl-methylsiloxane; Temperature programming: 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%.
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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside 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, extracting solution evaporate to dryness, residue add and are transferred in the separatory funnel after water makes dissolving, use ethyl acetate extraction; Add water washing, discard acetic acid ethyl fluid, merge water liquid, use water saturated n-butanol extraction; Extract the back with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins water eluting, discard water liquid, reuse 70% ethanol elution is collected eluent, evaporate to dryness, with dissolve with methanol and be transferred in the measuring bottle, add methanol to scale, shake up, filter with microporous filter membrane, 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 C
41H
68O
14Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with astragaloside C
41H
68O
14Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with astragaloside C
41H
68O
14Meter must not be less than 0.01mg/g;
(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of OV-17 fixative 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, sucking 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 standardize solution 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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue added and are transferred in the separatory 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, with water saturated n-butanol extraction 3-5 time, 20-60ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 20-60ml washing 1-2 time, n-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 eluting discards water liquid, reuse 70% ethanol 60-100ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter, promptly get need testing solution with 0.45 μ m microporous filter membrane; 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 C
41H
68O
14Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with astragaloside C
41H
68O
14Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with astragaloside C
41H
68O
14Meter must not be less than 0.01mg/g;
(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of OV-17 50% methyl-50% phenyl polysiloxane fixative 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, sucking 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:
Character: 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-coat to sepia, mildly bitter flavor Hui Tian;
For granule, product is that pale brown color is to auburn granule; 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 reflux, extract, 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 methanol, put reflux, extract, in the water-bath, reclaim methanol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separatory funnel, reuse water washing container, be transferred in the separatory funnel, use water saturated n-butanol extraction, extracting solution washes with water, divide and get n-butyl alcohol liquid, evaporate to dryness, the residue water dissolution is transferred on the neutral alumina post, use earlier the chloroform eluting, discard the chloroform eluent, the reuse methanol-eluted fractions, eluent is evaporate to dryness in water-bath, residue adds methanol makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer chromatography, draw need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 200-500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
(2) get capsule, tablet or granule, porphyrize, the reflux that adds diethyl ether is put coldly, filters, and discards filtrate, and medicinal residues add the methanol reflux, put coldly, filter, the filtrate evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Other gets the Herba Portulacae Grandiflorae control medicinal material, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is developing solvent, launch, take out, dry; Put under the 200-500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(3) get capsule, tablet or granule, porphyrize adds methanol, and reflux is put cold, filter, filtrate evaporate to dryness, residue add methanol and dissolve in right amount, are added on the neutral alumina post, use methanol-eluted fractions, collect eluent, evaporate to dryness, residue add methanol 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 layer chromatography, draw need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 the Fructus Ligustri Lucidi control medicinal material, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 an immobile phase with 5% phenyl-methylsiloxane; Temperature programming: 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 pertinent 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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside 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, extracting solution evaporate to dryness, residue add and are transferred in the separatory funnel after water makes dissolving, use ethyl acetate extraction; Add water washing, discard acetic acid ethyl fluid, merge water liquid, use water saturated n-butanol extraction; Extract the back with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins water eluting, discard water liquid, reuse 70% ethanol elution is collected eluent, evaporate to dryness, with dissolve with methanol and be transferred in the measuring bottle, add methanol to scale, shake up, filter with microporous filter membrane, 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 C
41H
68O
14Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with astragaloside C
41H
68O
14Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with astragaloside C
41H
68O
14Meter must not be less than 0.01mg/g;
(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of OV-17 fixative 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, sucking 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 standardize solution 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.
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:
Character: 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-coat to sepia, mildly bitter flavor Hui Tian;
For granule, product is that pale brown color is to auburn granule; It is sweet to distinguish the flavor of;
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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 2-6 hour, reclaim methanol to doing, residue adds 5% sodium hydroxide solution 1-10ml makes dissolving, goes in the separatory funnel 1-3 washing container of reuse 1-10ml moisture, be transferred in the separatory funnel, with water saturated n-butanol extraction 2-6 time, each 2-20ml merges n-butyl alcohol liquid, wash with water 1-3 time, each 1-10ml divides and gets n-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 eluting, discard the chloroform eluent earlier, reuse 70% methanol 10-40ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 0.5-2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 200-500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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, medicinal residues add methanol 10-40ml, reflux 10-50 minute, put cold, filter, filtrate evaporate to dryness, residue add methanol 0.5-2ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 0.5-2g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: acid=2-20: 1-9: 1-9 is developing solvent, launch, take out, dry; Put under the 200-500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(3) get capsule, tablet or granule 1-5g, porphyrize adds 50% methanol 10-50ml, reflux 0.5-2 hour, put coldly, filter, the filtrate evaporate to dryness, residue adds 50% methanol and dissolves in right amount, is added on 4g, 100～200 purpose neutral alumina posts, with 40% methanol 20-80ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5-2ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle 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 dehydrated alcohol 0.5-2ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 0.2-2g, shines medical material solution in pairs with legal system; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 an immobile phase with 5% phenyl-methylsiloxane; Temperature programming: 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%;
Other capsules of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the pertinent 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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue added and are transferred in the separatory 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, with water saturated n-butanol extraction 3-5 time, 20-60ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 20-60ml washing 1-2 time, n-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 eluting discards water liquid, reuse 70% ethanol 60-100ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter, promptly get need testing solution with 0.45 μ m microporous filter membrane; 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 C
41H
68O
14Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with astragaloside C
41H
68O
14Meter must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with astragaloside C
41H
68O
14Meter must not be less than 0.01mg/g;
(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of OV-17 50% methyl-50% phenyl polysiloxane fixative 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, sucking 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.
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 screening tests, and following experimentation is a preferred process of the present invention:
One, the research of Radix Astragali content assaying method
Relatively poor because of the astragaloside uv absorption, belong to end absorption, if detect with UV-detector, then its sensitivity is low, disturbs greatly, repeatability is relatively poor, especially for compound preparation, measures its content difficulty more.In recent years, the bibliographical information that uses the HPLC-ELSD method to measure Astragaloside content is arranged, its sensitivity, stability and repeatability 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 in the Milkvetch Root to chromatographic condition, and theoretical cam curve (N) press the calculating of astragaloside 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% sodium hydroxide alcoholic solution, extract the water bath method extracting solution in heating in water bath, add water, slight fever makes dissolving, puts the extraction that adds methylene chloride in the separatory funnel, abandon dichloromethane solution, the aqueous solution ether extraction is abandoned ether solution, the water saturated n-butanol extraction of aqueous solution, 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 methanol, shake up, filter with microporous filter membrane (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 methanol heating and refluxing extraction, the methanol solution evaporate to dryness, residue adds water, and slight fever makes dissolving, water liquid water saturation n-butanol extraction, n-butyl alcohol liquid washs with 2% potassium hydroxide solution, discards alkali liquor, the washing of reuse n-butyl alcohol saturation water discards water liquid, the n-butanol layer evaporate to dryness, residue adds methanol makes dissolving, and quantitatively is transferred in the measuring bottle, is diluted to scale with methanol, shake up, filter with microporous filter membrane (0.45 μ m), promptly.
Method 3: the product of getting it filled, porphyrize is got fine powder and added methanol, and is ultrasonic, pour out supernatant liquid filtering, it is ultrasonic that residue adds methanol, filters, and merges filtrate, evaporate to dryness, residue is dissolved in water, and uses dichloromethane extraction, water liquid water saturation n-butanol extraction, n-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 methanol, shake up, filter with microporous filter membrane (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 methanol, and placement is spent the night, and it is an amount of to add methanol again, reflux, extracting solution reclaims solvent and evaporate to dryness, and residue adds water, and slight fever makes dissolving, water liquid water saturation n-butanol extraction; N-butyl alcohol liquid washs with ammonia solution, discards ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, and residue adds water, slight fever makes dissolving, put cold, by D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), the water eluting discards water liquid, and reuse 30% ethanol elution discards eluent, continue and use 70% ethanol elution, collect eluent, evaporate to dryness, residue adds methanol makes dissolving, and quantitatively is transferred in the measuring bottle, is diluted to scale with methanol, shake up, filter with microporous filter membrane (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, extracting solution evaporate to dryness, residue add and are transferred in the separatory funnel after water makes dissolving, use ethyl acetate extraction; Acetic acid ethyl fluid adds water washing; Merge water liquid, use the water saturation n-butanol extraction; N-butanol extracting liquid washs with ammonia solution, discards ammoniacal liquor, n-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 eluting discards water liquid, reuse 70% ethanol elution is collected 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 microporous filter membrane (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 result of the test as can be known, employing method 5 preparation need testing solutions, astragaloside 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 standardize solution, and the impurity of sample disturbs bigger simultaneously, method four content with 30% washing with alcohol the time 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 the preparation method 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 fully.See the following form.
The investigation of extraction time
| Extraction time (h) | Sample weighting amount (g) | Content (mg/g) | Average content (mg/g) | RSD(％) |
| 3 | 1.0778 | 0.3746 | 0.3748 | 0.1 |
| 1.0716 | 0.3751 | |||
| 4 | 1.0116 | 0.4195 | 0.4193 | 0.1 |
| 1.0148 | 0.4191 | |||
| 6 | 1.0273 | 0.4160 | 0.4156 | 0.1 |
| 1.0327 | 0.4152 |
3, the investigation of n-butanol extraction number of times
6 parts of sample thiefs, take by weighing about 1g respectively, the accurate title, decide, preparation method 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 n-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 in the n-butanol extracting liquid of the 4th, and astragaloside has been less than 0.01% in the n-butyl alcohol liquid of the 5th.Therefore the extraction time with n-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
| Extraction time (inferior) | Sample weighting amount (g) | Content (mg/g) | Average content (mg/g) | RSD (％) |
| 2 | 1.0325 | 0.3249 | 0.3247 | 0.1 |
| 1.0374 | 0.3245 | |||
| 3 | 1.0624 | 0.3817 | 0.3816 | 0.0 |
| 1.0715 | 0.3815 | |||
| 4 | 1.0247 | 0.4159 | 0.4154 | 0.2 |
| 1.0281 | 0.4149 | |||
| 5 | 1.0138 | 0.4170 | 0.4166 | 0.2 |
| 1.0184 | 0.4161 |
(2) selection of mobile phase:
Mobile phase 1: the mixed solution with methanol, ethyl acetate solution different proportion is a mobile phase.
Mobile phase 2: the mixed solution with first alcohol and water different proportion is a mobile phase.
Mobile phase 3: the mixed solution with acetonitrile and water different proportion is a mobile phase.
The result: with acetonitrile: water=10-90: 90-10 is mobile phase; The negative sample chromatogram is at non-false positive peak, astragaloside position, and astragaloside separates fully (separating degree＞1.5) with close impurity peaks, and promptly astragaloside 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 methanol 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 chromatograph of liquid of stock solution drawn, measure its peak area, and be vertical coordinate with the natural logrithm (LNA) of peak area value A, the natural logrithm of sample size C (LNC) is an abscissa, getting regression equation is: LNA=1.5525LNC+16.324, r=0.9994.Natural logrithm (LNA) with peak area A is mapped to the natural logrithm (LNC) of sample size C, gets a straight line, and its result shows that the linear relationship of astragaloside is good in 0.4064～4.064 μ g scope.See the following form.
The astragaloside linear relationship is investigated
| LNC | LNA |
| -3.203 | 11.37410 |
| -2.5098 | 12.39351 |
| -1.81671 | 13.54332 |
| -1.41124 | 14.07012 |
| -0.90042 | 14.962210 |
| Regression equation LNA=1.5525LNC+16.324 | |
| Correlation coefficient r=0.9994 | |
(4) replica test:
Get 6 parts in same sample, every part of about 1g, the accurate title, decide, according to the preparation method 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 meansigma methods, and RSD is 0.5%, illustrates that repeatability is good.See the following form.
Replica test
| Sequence number | Sample weighting amount (g) | Astragaloside content (mg/g) | Meansigma methods (mg/g) | RSD (％) |
| 1 | 1.0216 | 0.4137 | 0.4118 | 0.5 |
| 2 | 1.0198 | 0.4132 | ||
| 3 | 1.0147 | 0.4122 | ||
| 4 | 1.0265 | 0.4131 | ||
| 5 | 1.0377 | 0.4080 | ||
| 6 | 1.0279 | 0.4105 |
(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 reference substance solution (0.2392mg/ml) 1ml that adds, according to the preparation method 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
| Sequence number | Sample sample weighting amount (g) | Addition (mg) | Record total amount (mg) | The response rate (%) | Meansigma methods (%) | RSD(％) |
| 1 | 0.5018 | 0.2392 | 0.4448 | 99.56 | 97.86 | 1.1 |
| 2 | 0.5197 | 0.2392 | 0.4451 | 96.61 | ||
| 3 | 0.5056 | 0.2392 | 0.4418 | 97.66 | ||
| 4 | 0.5183 | 0.2392 | 0.4455 | 97.02 | ||
| 5 | 0.5216 | 0.2392 | 0.4468 | 96.99 | ||
| 6 | 0.5048 | 0.2392 | 0.4454 | 99.30 |
(6) repeatability test
The product of getting it filled, take a sample according to the preparation method 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 meansigma methods 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 repeatability is good.See the following form.
The repeatability test
| Lot number | Laboratory group | Astragaloside content (mg/g) | Meansigma methods (mg/g) | Meansigma methods (mg/g) | RSD (％) |
| 01 | 1 | 0.4215 | 0.4212 | 0.4203 | 0.3 |
| 0.4209 | |||||
| 2 | 0.4198 | 0.4194 | |||
| 0.4190 | |||||
| 02 | 1 | 0.4113 | 0.4117 | 0.4127 | 0.3 |
| 0.4120 | |||||
| 2 | 0.4143 | 0.4137 | |||
| 0.4130 | |||||
| 03 | 1 | 0.3955 | 0.3940 | 0.3946 | 0.2 |
| 0.3924 | |||||
| 2 | 0.3958 | 0.3951 | |||
| 0.3944 |
(7) scope is investigated
According to the content limit of astragaloside in the medicine, its content is less than 1%, and the scope of investigation is ± 50%.The preparation of test sample is according to the preparation method 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 chromatograph of liquid, and the record peak area calculates content.See the following form.
Scope is investigated
| Sample | Sample weighting amount (g) | Content (mg/g) | Average content (mg/g) | RSD(％) |
| 1 | 0.5219 | 0.3884 | 0.3876 | 0.3 |
| 0.5248 | 0.3869 | |||
| 2 | 1.5016 | 0.3879 | 0.3868 | 0.4 |
| 1.5128 | 0.3858 |
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 in the working sample respectively.RSD% is 0.2%, and the result shows that sample solution is stable in 12h.
Stability experiment
| Sample injection time (hour) | Peak area value | Meansigma methods | RSD(％) |
| 0 | 1832496 | 1833200 | 0.2 |
| 2 | 1829616 | ||
| 4 | 1834687 |
| 6 | 1840015 | ||
| 8 | 1832649 | ||
| 12 | 1829734 |
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
| The post model | Separating degree | Measured value (mg/g) | Average content (mg/g) | RSD(％) |
| Diamonsil C18 5μ | 2.749 | 0.4235 | 0.4246 | 0.9 |
| Hypersil C18 5μ | 2.975 | 0.4215 | ||
| Kromasil C18 5μ | 2.872 | 0.4287 |
3. the variation of mobile 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
| Mobile phase ratio (acetonitrile-water) | Separating degree | Measured value (mg/g) | Average content (mg/g) | RSD(％) |
| 34∶66 | 2.263 | 0.4211 | 0.4215 | 0.1 |
| 35∶65 | 2.368 | 0.4219 | ||
| 36∶64 | 2.975 | 0.4215 |
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
| Column temperature (℃) | Separating degree | Measured value (mg/g) | Average content (mg/g) | RSD(％) |
| 30 | 2.797 | 0.4244 | 0.4234 | 0.4 |
| 35 | 2.784 | 0.4242 | ||
| 40 | 2.975 | 0.4215 |
(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 determination test in the sample
| Sequence number | Astragaloside content (mg/g) | Meansigma methods (mg/g) | RSD(％) |
| 1 | 0.3070 | 0.3043 | 1.3 |
| 0.3016 | |||
| 2 | 0.2831 | 0.2837 | 0.3 |
| 0.2843 | |||
| 3 | 0.3560 | 0.3558 | 0.1 |
| 0.3556 | |||
| 4 | 0.3830 | 0.3818 | 0.4 |
| 0.3806 | |||
| 5 | 0.3113 | 0.3101 | 0.5 |
| 0.3089 | |||
| 6 | 0.4235 | 0.4238 | 0.1 |
| 0.4240 | |||
| 7 | 0.2939 | 0.2950 | 0.5 |
| 0.2961 | |||
| 8 | 0.4157 | 0.4149 | 0.3 |
| 0.4141 | |||
| 9 | 0.3936 | 0.3949 | 0.5 |
| 0.3962 | |||
| 10 | 0.4136 | 0.4131 | 0.2 |
| 0.4126 |
Two, the research of cantharidin content assaying method
Because of cantharidin is anticancer active 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 medical material fine powder in the preparation of the present invention, and with general backflow, supersound 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 chloroform, put in the water-bath and reflux, put cold, filter, residue adds chloroform to be continued to reflux, and puts cold, filter, merging filtrate is put in the water-bath and is volatilized, residue dissolves with chloroform, 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 chloroform, put in the water-bath and reflux, put coldly, filter, residue adds a small amount of chloroform washing, and merging filtrate volatilizes naturally, with sodium chloride-sodium hydroxide solution washing residue, reuse hydrochloric acid is transferred pH to 1～2, filters, filtrate is used chloroform extraction, and chloroform solution volatilizes naturally, and residue adds the chloroform 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, supersound process is put cold, transfer pH to 1～2 with hydrochloric acid, sucking filtration washs residue with low amounts of water, merging filtrate is used chloroform extraction, and chloroform solution volatilizes naturally, residue adds the chloroform 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, sucking 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, the reuse chloroform is settled to 3ml, shakes 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 standardize solution 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 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 result of the test, 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 the preparation method 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 result of the test, 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 the preparation method 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 result of the test, 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 the preparation method 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 dichloromethane.See the following form.
Extract the comparison of solvent volume
| Solvent | Chloroform | Dichloromethane |
| Content (μ g/g) | 54.97 | 48.69 |
By the comparative test result, prove that chloroform can extract cantharidin fully, and the dichloromethane extraction rate 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 the preparation method 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 Mylabris 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 separating degree 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 standard 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
| Sample size (μ g) | The cantharidin peak area value |
| 0.0242 | 115501 |
| 0.0484 | 308234 |
| 0.0968 | 624302 |
| 0.121 | 756758 |
| 0.242 | 1488730 |
| 0.484 | 2963416 |
| Regression equation A=6127836.8097C+4767.9444 r=0.9998 | |
| The former point equation A=6142992.3864C of match r=0.9998 | |
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 the preparation method 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 meansigma methodss are 55.03 μ g/g, and RSD% is 1.63%, illustrate that repeatability is good.See the following form.
Replica test
| Sequence number | Sample weighting amount (g) | Cantharidin content (μ g/g) | Meansigma methods (μ g/g) | RSD(％) |
| 1 | 2.0016 | 54.16 | 55.03 | 1.63 |
| 2 | 2.0109 | 55.88 | ||
| 3 | 2.0101 | 54.47 | ||
| 4 | 2.0207 | 55.58 | ||
| 5 | 2.0058 | 54.06 | ||
| 6 | 2.0341 | 56.02 |
(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 response rate 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 sodium hydroxide mixed solution do not dissolve each other, cause having not ease for operation in extraction and the sucking 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 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 response rate 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
| Sequence number | Sample sample weighting amount (g) | Addition (μ g) | Record total amount (μ g) | The response rate (%) | Meansigma methods (%) | RSD％ |
| 1 | 1.0725 | 48.4 | 106.66 | 98.43 | 100.45 | 2.37 |
| 2 | 1.0652 | 48.4 | 106.9 | 99.76 | ||
| 3 | 1.0881 | 48.4 | 106.91 | 97.17 | ||
| 4 | 1.0881 | 48.4 | 109.32 | 102.15 | ||
| 5 | 1.1354 | 48.4 | 112.48 | 103.3 | ||
| 6 | 1.0797 | 48.4 | 108.73 | 101.89 |
(6) repeatability 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 at laboratory by different people respectively, the accurate respectively 2 μ L sample introductions of drawing, the record chromatograph, calculate content, the content meansigma methods 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 repeatability is good.See the following form.
The repeatability test
| Sequence number | Laboratory group | Sample weighting amount (g) | Cantharidin content (μ g/g) | Meansigma methods (μ g/g) | Meansigma methods (μ/g) | RSD ％ |
| 1 | A | 2.0833 | 78.68 | 78.73 | 77.74 | 1.81 |
| 2.0748 | 78.78 | |||||
| B | 2.0017 | 76.91 | 76.74 | |||
| 2.0011 | 76.57 | |||||
| 2 | A | 2.0661 | 53.49 | 53.68 | 52.92 | 2.03 |
| 2.0539 | 53.86 | |||||
| B | 2.0185 | 51.92 | 52.16 | |||
| 2.1322 | 52.41 | |||||
| 3 | A | 2.0049 | 56.32 | 55.88 | 55.80 | 0.22 |
| 2.0107 | 55.44 | |||||
| B | 2.0342 | 55.51 | 55.71 | |||
| 2.0050 | 55.91 |
(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 the preparation method 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 meansigma methodss 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
| Sequence number | Sample weighting amount (g) | Cantharidin content (μ g/g) | Meansigma methods (μ g/g) | RSD(％) |
| 1 | 1.0105 | 55.33 | 54.09 | 1.91 |
| 2 | 1.0126 | 54.30 | ||
| 3 | 2.9231 | 53.87 | ||
| 4 | 2.9007 | 52.86 |
(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
| Sample injection time (hour) | The cantharidin peak area value | Meansigma methods | RSD％ |
| 0 | 444649 |
| 2 | 441449 | 444789 | 0.45 |
| 4 | 445685 | ||
| 8 | 445447 | ||
| 12 | 446714 |
2. different gas chromatograpies, 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 the content meansigma methods, and RSD% is 0.22%.See the following form.
Different chromatographic columns
| The gas chromatograph model | Post model and chromatographic condition | Separating degree | Measured value (mg/g) | Average content (mg/g) | RSD ％ |
| GC-14C | A | 2.24 | 56.32 | 55.88 | 0.22 |
| 2.23 | 55.44 | ||||
| GC-2010 | B | 2.102 | 55.51 | 55.71 | |
| 1.638 | 55.91 |
Annotate: " A " is: chromatographic column: with carbowax-20M and methyl silicone rubber (SE-30) is fixative, 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 work station;
" 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 work station.
(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
| Sequence number | Sample weighting amount (g) | Cantharidin content (μ g/g) | Meansigma methods (μ g/g) | RSD(％) |
| 1 | 2.0146 | 49.11 | 49.08 | 0.10 |
| 2.0332 | 49.04 | |||
| 2 | 2.0061 | 51.97 | 52.36 | 1.07 |
| 2.0011 | 52.76 | |||
| 3 | 2.0351 | 44.87 | 45.09 | 0.69 |
| 2.0164 | 45.31 | |||
| 4 | 1.9841 | 82.39 | 82.10 | 0.50 |
| 2.0048 | 81.81 | |||
| 5 | 2.001 | 76.61 | 76.48 | 0.24 |
| 2.0073 | 76.35 | |||
| 6 | 2.0461 | 65.03 | 65.18 | 0.34 |
| 2.0215 | 65.34 | |||
| 7 | 2.0621 | 66.66 | 67.14 | 1.00 |
| 2.0304 | 67.61 | |||
| 8 | 2.0514 | 42.53 | 42.40 | 0.43 |
| 2.0693 | 42.27 | |||
| 9 | 2.0169 | 42.96 | 42.77 | 0.63 |
| 2.0304 | 42.58 | |||
| 10 | 2.1462 | 37.88 | 37.13 | 2.86 |
| 2.2133 | 36.38 | |||
| 11 | 2.0846 | 137.51 | 138.42 | 0.92 |
| 2.0599 | 139.32 | |||
| 12 | 2.0674 | 123.32 | 123.76 | 0.50 |
| 2.0519 | 124.19 |
Three, Radix Ginseng, Radix Astragali thin layer Study on Identification
Differentiate Radix Ginseng, Milkvetch Root in the preparation with ginsenoside Rg1's reference substance and astragaloside 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 reflux, extract, 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 methanol, put reflux, extract, in the water-bath, reclaim methanol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separatory funnel, reuse water washing container is transferred in the separatory funnel, uses water saturated n-butanol extraction, extracting solution washes with water, divides and gets n-butyl alcohol liquid, evaporate to dryness, the residue water dissolution is transferred to (neutral alumina 100～200 orders, 2g on the alumina column, dry column-packing, diameter 1cm, long 25cm), use earlier the chloroform eluting, discard the chloroform eluent, the reuse methanol-eluted fractions, eluent is evaporate to dryness in water-bath, residue adds methanol 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, supersound extraction discards chloroform solution, treats that chloroform waves to the greatest extent, add an amount of methanol, supersound extraction reclaims methanol to doing, and residue hydro-oxidation sodium solution makes dissolving, go in the separatory funnel, reuse water washing container is transferred in the separatory funnel, uses water saturated n-butanol extraction, extracting solution washes with water, divide and get n-butyl alcohol liquid, evaporate to dryness, residue water dissolution, be transferred to and use the chloroform eluting on the alumina column, eluent is evaporate to dryness in water-bath, and residue adds methanol 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.
Developing solvent is selected: respectively with the mixed solution of chloroform, ethyl acetate, methanol, water different proportion; The mixed solution of chloroform, methanol, water different proportion; The mixed solution of chloroform, ethyl acetate, water different proportion is developing solvent.
The thickness of lamellae 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 chloroform: methanol: water=5-50: 2-20: 0.5-10 is developing solvent, and its separating degree is good, and the speckle colour developing is clear, and negative control is noiseless, the method favorable reproducibility.With chloroform: ethyl acetate: methanol: water is developing solvent, and standing time is longer, operation inconvenience; And if the humidity of operating environment more also can cause the speckle separating effect bad, repeatability is relatively poor, by test of many times, think the lamellae 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 sulphuric acid; The thickness of lamellae 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 medical material content with the total amount of Radix Ginseng Re and Rg1 greater than 0.30% in the Radix Ginseng, adopting the point sample amount is test sample 8 μ l, and the speckle 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 developing solvent is: chloroform: methanol: water=13: 7: 2, more than the thickness 500 μ m of lamellae, the point sample amount is test sample 10 μ l, during reference substance 5 μ l, identification result the best.
Four, Herba Scutellariae Barbatae thin layer Study on Identification
Differentiate Herba Scutellariae Barbatae medical material in the preparation with the Herba Scutellariae Barbatae 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 medicinal residues add methanol, and reflux is put coldly, filters, and filtrate evaporate to dryness, residue add methanol makes dissolving, as need testing solution; The negative test liquid that lacks Herba Scutellariae Barbatae with the method preparation.
Need testing solution preparation method two: the product of getting it filled, porphyrize adds chloroform, and supersound extraction filters, and discards filtrate, and medicinal residues add methanol, and supersound extraction filters, and filtrate evaporate to dryness, residue add methanol makes dissolving, as need testing solution; The negative test liquid that lacks Herba Scutellariae Barbatae with the method preparation.
Developing solvent is selected: respectively with the mixed solution of toluene, Ethyl formate, formic acid different proportion; The mixed solution of cyclohexane extraction, ethyl acetate different proportion; The mixed solution of benzene and Ethyl formate different proportion; The mixed solution of cyclohexane extraction, formic acid different proportion is developing solvent.
The result: employing method one preparation need testing solution, with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is developing solvent, and its separating degree is good, and the speckle colour developing is clear, and negative control is noiseless, the method favorable reproducibility, specificity is strong.Best developing solvent is: toluene: Ethyl formate: formic acid=9: 5: 3.
Five, Fructus Corni thin layer Study on Identification
Method one: the thing of getting it filled, porphyrize adds ethyl acetate, and supersound process filters, and filtrate evaporate to dryness, residue add dehydrated alcohol makes dissolving, as need testing solution; The negative test liquid that lacks Fructus Corni with the method preparation.Other gets the ursolic acid reference substance and adds solution that dehydrated alcohol is mixed with 1mg/ml product solution in contrast.With toluene: ethyl acetate: formic acid=20: 4: 0.5 is developing solvent, 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 methanol, and reflux filters, and discards filtrate.Residue adds ethyl acetate, and supersound process filters, and filtrate evaporate to dryness, residue add dehydrated alcohol makes dissolving, as need testing solution; The negative test liquid that lacks Fructus Corni with the method preparation.Get the Fructus Corni control medicinal material, be equipped with control medicinal material solution with legal system.With cyclohexane extraction: chloroform: ethyl acetate=20: 5: 8 is developing solvent, 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 methanol, and supersound process filters, the filtrate evaporate to dryness, residue adds dehydrated alcohol: the mixed solution of chloroform=3: 2 makes dissolving, as need testing solution; The negative test liquid that lacks Fructus Corni with the method preparation.Other gets the Fructus Corni control medicinal material, is equipped with control medicinal material solution with legal system.With cyclohexane extraction: acetone: ethyl acetate=10: 4: 2 is developing solvent, 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 Fructus Corni and the Fructus Ligustri Lucidi, can cause negative the interference, therefore consideration selects for use in the Fructus Corni distinctive loganin to differentiate.
Differentiate Fructus Corni medical 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% methanol, reflux is put coldly, filters, filtrate evaporate to dryness, residue add 50% methanol 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 methanol makes dissolving, as need testing solution; The negative test liquid that lacks Fructus Corni with the method preparation.
Need testing solution preparation method two: the thing of getting it filled, grind well, add 50% methanol, supersound process is put coldly, filters, filtrate evaporate to dryness, residue add 50% methanol 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 methanol makes dissolving, as need testing solution; The negative sample solution that lacks Fructus Corni with the method preparation.
Developing solvent is selected: respectively with the mixed solution of toluene, ethyl acetate, formic acid different proportion;
The mixed solution of toluene, acetone, dehydrated alcohol, dense ammonia different proportion;
The mixed solution of toluene, acetone, dehydrated alcohol, formic acid different proportion;
The mixed solution of cyclohexane extraction, acetone, ethyl acetate different proportion;
The mixed solution of cyclohexane extraction, chloroform, ethyl acetate different proportion is developing solvent.
The result: employing method one preparation need testing solution, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, and its separating degree is good, and the speckle 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 developing solvent is: toluene: acetone: dehydrated alcohol: formic acid=4: 16: 2: 0.5.Adopt first three methods that Fructus Corni is carried out thin layer and differentiate that its identification result separating degree is bad, the speckle colour developing is unintelligible, and feminine gender has interference.
Six, Fructus Ligustri Lucidi thin layer Study on Identification
Differentiate Fructus Ligustri Lucidi medical material in the preparation with the Fructus Ligustri Lucidi control medicinal material:
Need testing solution preparation method one: the thing of getting it filled, grind well, add methanol, reflux is put coldly, filters, the filtrate evaporate to dryness, residue adds dehydrated alcohol: the mixed solution of chloroform=3: 2 makes dissolving, as need testing solution; The negative test liquid that lacks Fructus Ligustri Lucidi with the method preparation.
Need testing solution preparation method two: the thing of getting it filled, porphyrize decocts with water, and filters, and filtrate concentrates, and transfers pH to 2～3 with dilute 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 Fructus Ligustri Lucidi with the method preparation.
Need testing solution preparation method three: the thing of getting it filled, porphyrize decocts with water, and filters the filtrate evaporate to dryness.Residue adds dehydrated alcohol: the mixed solution of chloroform=3: 2 makes dissolving, as need testing solution; The negative test liquid that lacks Fructus Ligustri Lucidi with the method preparation.
Need testing solution preparation method four: the thing of getting it filled, porphyrize adds the methanol reflux, filters, and discards filtrate, and residue adds ethyl acetate, and is ultrasonic, filters, and filtrate evaporate to dryness, residue add dehydrated alcohol makes dissolving, as need testing solution; The negative test liquid that lacks Fructus Ligustri Lucidi 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 dehydrated alcohol makes dissolving, as need testing solution; Lack the negative test liquid of Fructus Ligustri Lucidi with the method preparation.
Developing solvent is selected: respectively with the mixed solution of cyclohexane extraction, acetone, ethyl acetate different proportion;
The mixed solution of toluene, ethyl acetate, formic acid different proportion;
The mixed solution of petroleum ether, ethyl acetate, formic acid different proportion is developing solvent.
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 developing solvent, and its separating degree is good, and the speckle colour developing is clear, and negative control is noiseless, and the method favorable reproducibility differentiates that specificity is strong.Best developing solvent is: toluene: ethyl acetate: formic acid=20: 4: 0.5.
Seven, the thin layer Study on Identification of Fel Ursi powder
Fel Ursi 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 dilute hydrochloric acid to pH 2～3, goes in the separatory funnel, uses ethyl acetate extraction, merge extractive liquid,, and evaporate to dryness, residue add ethanol makes dissolving, as need testing solution; The negative test liquid that lacks Fel Ursi 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 dilute hydrochloric acid to pH 2～3, goes in the separatory funnel, uses ethyl acetate extraction, merge extractive liquid,, and evaporate to dryness, residue add ethanol makes dissolving, as need testing solution; The negative test liquid that lacks Fel Ursi powder with the method preparation.
Method three: the thing of getting it filled adds 10% sodium hydroxide, ultrasonic dissolving, 120 ℃ of heating hydrolysis of making.Put coldly, filter, filtrate drips dilute hydrochloric acid to pH 2～3, goes in the separatory funnel, and with ethyl acetate extraction twice, merge extractive liquid,, evaporate to dryness, residue add ethanol makes dissolving, as need testing solution; The negative test liquid that lacks Fel Ursi 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% sodium hydroxide in heating in water bath hydrolysis (replenishing the water of evaporation at any time).Put coldly, drip dilute hydrochloric acid, go in the separatory funnel, use ethyl acetate extraction to pH 2～3, merge extractive liquid,, evaporate to dryness, residue add ethanol makes dissolving, as need testing solution; The negative test liquid that lacks Fel Ursi 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% sodium hydroxide and is transferred in the appropriate containers, hydrolysis to the water-bath.Put coldly, dripping hydrochloric acid goes in the separatory funnel to PH 2～3, uses ethyl acetate extraction, merge extractive liquid,, and evaporate to dryness, residue add methanol makes dissolving, as need testing solution; The negative test liquid that lacks Fel Ursi powder with the method preparation.
Developing solvent is selected: respectively with the mixed solution of isobutyltrimethylmethane., ether, glacial acetic acid, n-butyl alcohol, water different proportion; The mixed solution of isobutyltrimethylmethane., ethyl acetate, glacial acetic acid different proportion is developing solvent.
Result: respectively with 10% ethanol solution of sulfuric acid, 10% phosphomolybdic acid ethanol solution colour developing, with the corresponding position of reference substance (ursodesoxycholic acid, chenodeoxycholic acid, Hyodeoxycholic Acid and cholic acid mix reference substance) on, speckle is unintelligible, and other impurity colors are very dark, disturbs bigger.Observe under fluorescence, impurity is also very big to main speckle influence.
The discrimination method of Fel Ursi 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 Fel Ursi 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 treatment process complexity, easily causes damage; In addition, oral formulations taste of 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 Fel Ursi powder.
Eight, the inspection method research of chloroform residual quantity
The Mylabris medical 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 work station; 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 an analytical pure.
(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-methylsiloxane is an immobile phase); Temperature programming: 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 separating degree 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 processing method 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 abscissa with reference substance solution, is that vertical coordinate 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
| Chloroform reference substance concentration (mg/ml) | The chloroform peak area |
| 0.0052272 | 321.6 |
| 0.008712 | 519.7 |
| 0.017424 | 883.2 |
| 0.052272 | 2474.8 |
| 0.17424 | 8969.3 |
| 0.34848 | 17769.8 |
| Regression equation: y=51031.5714x-0.8098 r=0.9999 | |
| Match is to the equation of crossing initial point: y=51028.4023x | |
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
| Numbering | Peak area | Average peak area | RSD％ |
| 1 | 871.6 | 889.3 | 2.2 |
| 2 | 881.6 | ||
| 3 | 901.3 | ||
| 4 | 874.2 | ||
| 5 | 883.2 | ||
| 6 | 923.8 |
(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 prescribed limit.The result shows and has good stability.See the following form:
Study on the stability
| Time | Peak area | Average peak area | RSD％ |
| 0 hour | 754.0 | 725.3 | 5.4 |
| 2 hours | 764.0 | ||
| 4 hours | 734.0 | ||
| 8 hours | 714.5 | ||
| 12 hours | 732.1 |
(8) repeatability is investigated
The FUFANG BANMAO JIAONANG 1.0g that gets the preparation method 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 response rate.The FUFANG BANMAO JIAONANG 1.0g that gets the preparation method 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
| Numbering | Sample weighting amount (g) | Former content | Addition (mg) | Record peak area | The amount of recording (mg) | Response rate % | Average recovery rate % | RSD％ |
| 1 | 1.1265 | Do not detect | 0.17424 | 836.9 | 0.16968 | 97.4 | 98.3 | 3.0 |
| 2 | 1.0045 | Do not detect | 0.17424 | 894.9 | 0.18144 | 104.1 | ||
| 3 | 1.0841 | Do not detect | 0.17424 | 842.8 | 0.17087 | 98.1 | ||
| 4 | 1.0219 | Do not detect | 0.17424 | 839.6 | 0.17023 | 97.7 | ||
| 5 | 1.0441 | Do not detect | 0.17424 | 826.0 | 0.16747 | 96.1 | ||
| 6 | 1.0021 | Do not detect | 0.17424 | 830.6 | 0.16840 | 96.6 |
(10) repeatability 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 repeatability 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 repeatability 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 Herba Scutellariae Barbatae of having set up cantharidin in the Radix Astragali and the Mylabris, Fructus Corni, the thin layer of Fructus Ligustri Lucidi is differentiated project, increased the inspection of chloroform residual quantity, and the thin layer discrimination method of Radix Ginseng and the Radix Astragali two flavor medical materials improved, thickness to developing solvent and lamellae is selected, optimized discrimination method, its precision height, favorable reproducibility, good stability, response rate 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.
The specific 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 FUFANG BANMAO JIAONANG agent comprises:
Character: 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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 4 hours, reclaim methanol to doing, residue adds 5% sodium hydroxide solution 5ml makes dissolving, goes in the separatory funnel 2 washing containers of reuse 5ml moisture, be transferred in the separatory funnel, with 4 (10ml of water saturated n-butanol extraction, 5ml, 5ml, 5ml), merge n-butyl alcohol liquid, wash with water 2 times, each 5ml divides and gets n-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 eluting, discards the chloroform eluent earlier, reuse 70% methanol 25ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 1ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 1mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: methanol: lower floor's solution of water=13: 7: 2 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 365nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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 medicinal residues add methanol 20ml, and reflux 30 minutes is put coldly, filters, and filtrate evaporate to dryness, residue add methanol 1ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 1g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=9: 5: 3 is developing solvent, launch, take out, dry; Put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
(3) get capsule content 3g, add 50% methanol 30ml, reflux 1 hour, put coldly, filter the filtrate evaporate to dryness, residue adds 50% methanol and dissolves in right amount, be added on the neutral alumina post (100～200 orders, 4g) on, with 40% methanol 50ml eluting, collect eluent, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=4: 16: 2: 0.5 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
(4) get capsule content 3g, add ethyl acetate 20ml, ultrasonic 15min filters, and filtrate evaporate to dryness, residue add dehydrated alcohol 1ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 0.5g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=20: 4: 0.5 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 an immobile phase); Temperature programming: 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.
Algoscopy 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 pertinent 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 filler, and with acetonitrile: water=36: 64 is mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue adds and is transferred in the separatory 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, with water saturated n-butanol extraction 4 times, 40ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 40ml washing 1 time, n-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 eluting discards water liquid, reuse 70% ethanol 80ml eluting is collected eluent, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter with 0.45 μ m microporous filter membrane, 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 (C
41H
68O
14) meter, must not be less than 0.12mg/g.
(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of OV-17 (50% methyl-50% phenyl polysiloxane) fixative 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, sucking 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:
Character: product is a Film coated tablets, removes to show yellow green behind the film-coat 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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 3 hours, reclaim methanol to doing, residue adds 5% sodium hydroxide solution 3ml makes dissolving, goes in the separatory funnel, 2 washing containers of reuse 3ml moisture are transferred in the separatory funnel, with water saturated n-butanol extraction 3 times, each 10ml merges n-butyl alcohol liquid, washes with water 2 times, each 3ml divides and gets n-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 eluting, discards the chloroform eluent earlier, reuse 70% methanol 20ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 0.8ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 0.8mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: methanol: lower floor's solution of water=20: 10: 5 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 300nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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 medicinal residues add methanol 25ml, and reflux 20 minutes is put coldly, filters, and filtrate evaporate to dryness, residue add methanol 0.8ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 0.8g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=10: 5: 5 is developing solvent, launch, take out, dry; Put under the 300nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
(3) get tablet 2g, porphyrize adds 50% methanol 20ml, reflux 1.2 hours is put coldly, filters, filtrate evaporate to dryness, residue add 50% methanol and dissolve in right amount, are added on neutral alumina post (100～200 orders, 4g), with 40% methanol 35ml eluting, collect eluent, evaporate to dryness, residue adds methanol 0.8ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 0.8mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=5: 25: 5: 0.5 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
(4) get tablet 2g, porphyrize adds ethyl acetate 25ml, and ultrasonic 20min filters, and filtrate evaporate to dryness, residue add dehydrated alcohol 0.8ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 1g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=30: 5: 0.5 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 siloxanes is an immobile phase); Temperature programming: 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 pertinent 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 filler, and with acetonitrile: water=50: 50 is mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue adds and is transferred in the separatory 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, with water saturated n-butanol extraction 4 times, 30ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 30ml washing 2 times, n-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 eluting discards water liquid, reuse 70% ethanol 70ml eluting is collected eluent, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter with 0.45 μ m microporous filter membrane, 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 (C
41H
68O
14) meter, must not be less than 0.08mg/g.
(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of OV-17 (50% methyl-50% phenyl polysiloxane) fixative 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, sucking 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:
Character: product is that pale brown color is to auburn granule; 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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 5 hours, reclaim methanol to doing, residue adds 5% sodium hydroxide solution 7ml makes dissolving, goes in the separatory funnel, 2 washing containers of reuse 7ml moisture are transferred in the separatory funnel, with water saturated n-butanol extraction 5 times, each 15ml merges n-butyl alcohol liquid, washes with water 2 times, each 7ml divides and gets n-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 eluting, discards the chloroform eluent earlier, reuse 70% methanol 30ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 1.5ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 1.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: methanol: lower floor's solution of water=40: 15: 8 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 400nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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 medicinal residues add methanol 30ml, and reflux 40 minutes is put coldly, filters, and filtrate evaporate to dryness, residue add methanol 1.5ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 1.5g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=15: 7: 4 is developing solvent, launch, take out, dry; Put under the 400nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
(3) get granule 4g, porphyrize adds 50% methanol 40ml, reflux 1.5 hours is put coldly, filters, filtrate evaporate to dryness, residue add 50% methanol and dissolve in right amount, are added on neutral alumina post (100～200 orders, 4g), with 40% methanol 65ml eluting, collect eluent, evaporate to dryness, residue adds methanol 1.5ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 1.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=7: 40: 8: 0.7 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
(4) get granule 4g, porphyrize adds ethyl acetate 30ml, and ultrasonic 25min filters, and filtrate evaporate to dryness, residue add dehydrated alcohol 1.5ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 1.5g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=40: 7: 0.8 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 siloxanes is an immobile phase); Temperature programming: 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 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 pertinent 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 filler, and with acetonitrile: water=60: 40 is mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue adds and is transferred in the separatory 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, with water saturated n-butanol extraction 4 times, 50ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 50ml washing 2 times, n-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 eluting discards water liquid, reuse 70% ethanol 90ml eluting is collected eluent, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter with 0.45 μ m microporous filter membrane, 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 (C
41H
68O
14) meter, must not be less than 0.01mg/g.
(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of OV-17 (50% methyl-50% phenyl polysiloxane) fixative 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, sucking 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
120
4) be 0.003-0.018mg/g.
Embodiments of the invention 4: the method for quality control of compound mylabris preparation can comprise:
Character: 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-coat to sepia, mildly bitter flavor Hui Tian;
For granule, product is that pale brown color is to auburn granule; 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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 2 hours, reclaim methanol to doing, residue adds 5% sodium hydroxide solution lml makes dissolving, goes in the separatory funnel reuse 1ml water washing container, be transferred in the separatory funnel, with water saturated n-butanol extraction 2 times, each 2ml merges n-butyl alcohol liquid, wash with water 1 time, each 1ml divides and gets n-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 eluting, discard the chloroform eluent earlier, reuse 70% methanol 10ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 0.5ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 0.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: methanol: lower floor's solution of water=5: 20: 0.5 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 200nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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, medicinal residues add methanol 10ml, and reflux 10 minutes is put cold, filter, filtrate evaporate to dryness, residue add methanol 0.5ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 0.5g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=2: 9: 1 is developing solvent, launch, take out, dry; Put under the 200nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 pertinent regulations under capsule, tablet or the granule item.
Assay: cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of 0V-17 (50% methyl-50% phenyl polysiloxane) fixative is 1.5%, carrier Shimalite W (AW-DMCS) 80-100 order; 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 the 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, sucking 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:
Character: 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-coat to sepia, mildly bitter flavor Hui Tian;
For granule, product is that pale brown color is to auburn granule; It is sweet to distinguish the flavor of.
Differentiate: (1) gets capsule, tablet or granule 5g, and porphyrize adds 50% methanol 10ml, reflux 0.5 hour is put coldly, filters, the filtrate evaporate to dryness, residue adds 50% methanol and dissolves in right amount, is added on the neutral alumina post, with 40% methanol 20ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1: 50: 0.5: 0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle 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 dehydrated alcohol 0.5ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 0.2g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10: 9: 0.1 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 pertinent regulations under capsule, tablet or the granule item.
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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue add and are transferred in the separatory 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, with water saturated n-butanol extraction 3 times, 20ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 20ml washing 1 time, n-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 eluting discards water liquid, reuse 70% ethanol 60ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter, promptly get need testing solution with 0.45 μ m microporous filter membrane; 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 (C
41H
68O
14) meter, must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with astragaloside (C
41H
68O
14) meter, must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with astragaloside (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:
Character: 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-coat to sepia, mildly bitter flavor Hui Tian;
For granule, product is that pale brown color is to auburn granule; It is sweet to distinguish the flavor of.
Differentiate: get capsule, tablet or granule 5g, porphyrize adds ethyl acetate 40ml, and ultrasonic 30min filters, and filtrate evaporate to dryness, residue add dehydrated alcohol 2ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 2g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=50: 1: 0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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-methylsiloxane is an immobile phase); Temperature programming: 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%.
Other capsules of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the pertinent regulations under capsule, tablet or the granule item.
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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue add and are transferred in the separatory 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, with water saturated n-butanol extraction 5 times, 60ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 60ml washing 2 times, n-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 eluting discards water liquid, reuse 70% ethanol 100ml eluting is collected eluent, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter with 0.45 μ m microporous filter membrane, 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 (C
41H
68O
14) meter, must not be less than 0.08mg/g; Contain the Radix Astragali in the tablet with astragaloside (C
41H
68O
14) meter, must not be less than 0.08mg/g; Contain the Radix Astragali in the granule with astragaloside (C
41H
68O
14) meter, must not be less than 0.01mg/g.
(2) cantharidin shines gas chromatography determination: glass column 1-2m * 3mm id, and the stain amount that is coated with of OV-17 (50% methyl-50% phenyl polysiloxane) fixative 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, sucking 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:
Character: 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-coat to sepia, mildly bitter flavor Hui Tian;
For granule, product is that pale brown color is to auburn granule; It is sweet to distinguish the flavor of.
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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 6 hours, reclaim methanol to doing, residue adds 5% sodium hydroxide solution 10ml makes dissolving, goes in the separatory funnel 3 washing containers of reuse 10ml moisture, be transferred in the separatory funnel, with water saturated n-butanol extraction 6 times, each 20ml merges n-butyl alcohol liquid, wash with water 3 times, each 10ml divides and gets n-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 eluting, discard the chloroform eluent earlier, reuse 70% methanol 40ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: methanol: lower floor's solution of water=50: 2: 10 is developing solvent (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 speckle colour developing at 105 ℃, puts under the 500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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, medicinal residues add methanol 40ml, and reflux 50 minutes is put cold, filter, filtrate evaporate to dryness, residue add methanol 2ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 2g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=20: 1: 9 is developing solvent, launch, take out, dry; Put under the 500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
(3) get capsule, tablet or granule 5g, porphyrize adds 50% methanol 50ml, reflux 2 hours is put coldly, filters, the filtrate evaporate to dryness, residue adds 50% methanol and dissolves in right amount, is added on the neutral alumina post, with 40% methanol 80ml eluting, collect eluent, evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive (point becomes band) with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=9: 5: 10: 0.1 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle 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-methylsiloxane is an immobile phase); Temperature programming: 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%.
Other capsules of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the pertinent regulations under capsule, tablet or the granule item.
Claims (11)
1. the method for quality control of a compound cantharidin oral preparations, described oral formulations is capsule, tablet or granule, it is characterized in that: described method of quality control mainly comprise in character, discriminating, inspection and the assay project partly or entirely; Wherein differentiate the thin layer chromatography discriminating that comprises Radix Ginseng, the Radix Astragali, Herba Scutellariae Barbatae, Fructus Corni and Fructus Ligustri Lucidi 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 Mylabris.
2. according to the method for quality control of the described compound cantharidin oral preparations of claim 1, it is characterized in that: the discrimination method of the Radix Ginseng and the Radix Astragali is to be contrast with ginsenoside Rg1's reference substance and astragaloside reference substance, and with chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is the thin layer chromatography of developing solvent; The discrimination method of Herba Scutellariae Barbatae is to be contrast with the Herba Scutellariae Barbatae control medicinal material, and with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is the thin layer chromatography of developing solvent; The discrimination method of Fructus Corni is to be contrast with the loganin reference substance, and with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is the thin layer chromatography of developing solvent; The discrimination method of Fructus Ligustri Lucidi is to be contrast with the Fructus Ligustri Lucidi control medicinal material, and with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is the thin layer chromatography of developing solvent; The inspection method of chloroform residual quantity is to be the gas chromatography of contrast with the chloroform reference substance; The content assaying method of the Radix Astragali is to be contrast with the astragaloside reference substance, is the high performance liquid chromatography of mobile phase with acetonitrile: water=10-90: 90-10; The content assaying method of contained cantharidin is to be the gas chromatography of contrast with the cantharidin reference substance in the Mylabris.
3. according to the method for quality control of claim 1 or 2 described compound cantharidin oral preparations, it is characterized in that: 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 reflux, extract, 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 methanol, put reflux, extract, in the water-bath, reclaim methanol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separatory funnel, reuse water washing container, be transferred in the separatory funnel, use water saturated n-butanol extraction, extracting solution washes with water, divide and get n-butyl alcohol liquid, evaporate to dryness, the residue water dissolution is transferred on the neutral alumina post, use earlier the chloroform eluting, discard the chloroform eluent, the reuse methanol-eluted fractions, eluent is evaporate to dryness in water-bath, residue adds methanol makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer chromatography, draw need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts under the 200-500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
(2) get capsule, tablet or granule, porphyrize, the reflux that adds diethyl ether is put coldly, filters, and discards filtrate, and medicinal residues add the methanol reflux, put coldly, filter, the filtrate evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Other gets the Herba Portulacae Grandiflorae control medicinal material, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is developing solvent, launch, take out, dry; Put under the 200-500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(3) get capsule, tablet or granule, porphyrize adds methanol, and reflux is put cold, filter, filtrate evaporate to dryness, residue add methanol and dissolve in right amount, are added on the neutral alumina post, use methanol-eluted fractions, collect eluent, evaporate to dryness, residue add methanol 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 layer chromatography, draw need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 the Fructus Ligustri Lucidi control medicinal material, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
4. according to the method for quality control of the described compound cantharidin oral preparations of claim 3, it is characterized in that: 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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 2-6 hour, reclaim methanol to doing, residue adds 5% sodium hydroxide solution 1-10ml makes dissolving, goes in the separatory funnel 1-3 washing container of reuse 1-10ml moisture, be transferred in the separatory funnel, with water saturated n-butanol extraction 2-6 time, each 2-20ml merges n-butyl alcohol liquid, wash with water 1-3 time, each 1-10ml divides and gets n-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 eluting, discard the chloroform eluent earlier, reuse 70% methanol 10-40ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 0.5-2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to speckle colour developing at 105 ℃, puts under the 200-500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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, medicinal residues add methanol 10-40ml, reflux 10-50 minute, put cold, filter, filtrate evaporate to dryness, residue add methanol 0.5-2ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 0.5-2g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is developing solvent, launch, take out, dry; Put under the 200-500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(3) get capsule, tablet or granule 1-5g, porphyrize adds 50% methanol 10-50ml, reflux 0.5-2 hour, put coldly, filter, the filtrate evaporate to dryness, residue adds 50% methanol and dissolves in right amount, is added on 4g, 100～200 purpose neutral alumina posts, with 40% methanol 20-80ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5-2ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle 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 dehydrated alcohol 0.5-2ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 0.2-2g, shines medical material solution in pairs with legal system; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
5. according to the method for quality control of claim 1 or 2 described compound cantharidin oral preparations, 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 an immobile phase with 5% phenyl-methylsiloxane; Temperature programming: 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%.
6. according to the method for quality control of the described compound cantharidin oral preparations of claim 5, 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 an immobile phase with 5% phenyl-methylsiloxane; Temperature programming: 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%.
7. according to the method for quality control of claim 1 or 2 described compound cantharidin oral preparations, it is characterized in that: 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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside 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, extracting solution evaporate to dryness, residue add and are transferred in the separatory funnel after water makes dissolving, use ethyl acetate extraction; Add water washing, discard acetic acid ethyl fluid, merge water liquid, use water saturated n-butanol extraction; Extract the back with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins water eluting, discard water liquid, reuse 70% ethanol elution is collected eluent, evaporate to dryness, with dissolve with methanol and be transferred in the measuring bottle, add methanol to scale, shake up, filter with microporous filter membrane, 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, must not be less than 0.08mg/g in astragaloside C41H68O14; Contain the Radix Astragali in the tablet in astragaloside C41H68O14, must not be less than 0.08mg/g; Contain the Radix Astragali in the granule in astragaloside C41H68O14, 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 fixative 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, sucking 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 standardize solution 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, containing cantharidin C10H12O4 in the capsule is 0.025-0.180mg/g; Containing cantharidin C10H12O4 in the tablet is 0.025-0.180mg/g; Containing cantharidin C10H12O4 in the granule is 0.003-0.018mg/g.
8. according to the method for quality control of the described compound cantharidin oral preparations of claim 7, it is characterized in that: 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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue added and are transferred in the separatory 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, with water saturated n-butanol extraction 3-5 time, 20-60ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 20-60ml washing 1-2 time, n-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 eluting discards water liquid, reuse 70% ethanol 60-100ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter, promptly get need testing solution with 0.45 μ m microporous filter membrane; 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, must not be less than 0.08mg/g in astragaloside C41H68O14; Contain the Radix Astragali in the tablet in astragaloside C41H68O14, must not be less than 0.08mg/g; Contain the Radix Astragali in the granule in astragaloside C41H68O14, 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 fixative 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, sucking 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, containing cantharidin C10H12O4 in the capsule is 0.025-0.180mg/g; Containing cantharidin C10H12O4 in the tablet is 0.025-0.180mg/g; Containing cantharidin C10H12O4 in the granule is 0.003-0.018mg/g.
9. according to the method for quality control of the described compound cantharidin oral preparations of claim 1, it is characterized in that: described method of quality control comprises:
Character: 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-coat to sepia, mildly bitter flavor Hui Tian;
For granule, product is that pale brown color is to auburn granule; 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 reflux, extract, 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 methanol, put reflux, extract, in the water-bath, reclaim methanol to doing, residue hydro-oxidation sodium solution makes dissolving, goes in the separatory funnel, reuse water washing container, be transferred in the separatory funnel, use water saturated n-butanol extraction, extracting solution washes with water, divide and get n-butyl alcohol liquid, evaporate to dryness, the residue water dissolution is transferred on the neutral alumina post, use earlier the chloroform eluting, discard the chloroform eluent, the reuse methanol-eluted fractions, eluent is evaporate to dryness in water-bath, residue adds methanol makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds dissolve with methanol, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer chromatography, draw need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts under the 200-500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
(2) get capsule, tablet or granule, porphyrize, the reflux that adds diethyl ether is put coldly, filters, and discards filtrate, and medicinal residues add the methanol reflux, put coldly, filter, the filtrate evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Other gets the Herba Portulacae Grandiflorae control medicinal material, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is developing solvent, launch, take out, dry; Put under the 200-500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(3) get capsule, tablet or granule, porphyrize adds methanol, and reflux is put cold, filter, filtrate evaporate to dryness, residue add methanol and dissolve in right amount, are added on the neutral alumina post, use methanol-eluted fractions, collect eluent, evaporate to dryness, residue add methanol 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 layer chromatography, draw need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 the Fructus Ligustri Lucidi control medicinal material, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 an immobile phase with 5% phenyl-methylsiloxane; Temperature programming: 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%;
Other capsules of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the pertinent 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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside 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, extracting solution evaporate to dryness, residue add and are transferred in the separatory funnel after water makes dissolving, use ethyl acetate extraction; Add water washing, discard acetic acid ethyl fluid, merge water liquid, use water saturated n-butanol extraction; Extract the back with the ammonia solution washing, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue adds water makes dissolving in right amount, is transferred on the D101 type macroporous adsorptive resins water eluting, discard water liquid, reuse 70% ethanol elution is collected eluent, evaporate to dryness, with dissolve with methanol and be transferred in the measuring bottle, add methanol to scale, shake up, filter with microporous filter membrane, 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, must not be less than 0.08mg/g in astragaloside C41H68O14; Contain the Radix Astragali in the tablet in astragaloside C41H68O14, must not be less than 0.08mg/g; Contain the Radix Astragali in the granule in astragaloside C41H68O14, 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 fixative 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, sucking 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 standardize solution 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, containing cantharidin C10H12O4 in the capsule is 0.025-0.180mg/g; Containing cantharidin C10H12O4 in the tablet is 0.025-0.180mg/g; Containing cantharidin C10H12O4 in the granule is 0.003-0.018mg/g.
10. according to the method for quality control of the described compound cantharidin oral preparations of claim 9, it is characterized in that: described method of quality control comprises:
Character: 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-coat to sepia, mildly bitter flavor Hui Tian;
For granule, product is that pale brown color is to auburn granule; It is sweet to distinguish the flavor of;
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 reflux, extract, 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 methanol, put in the water-bath reflux, extract, 2-6 hour, reclaim methanol to doing, residue adds 5% sodium hydroxide solution 1-10ml makes dissolving, goes in the separatory funnel 1-3 washing container of reuse 1-10ml moisture, be transferred in the separatory funnel, with water saturated n-butanol extraction 2-6 time, each 2-20ml merges n-butyl alcohol liquid, wash with water 1-3 time, each 1-10ml divides and gets n-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 eluting, discard the chloroform eluent earlier, reuse 70% methanol 10-40ml eluting, eluent is evaporate to dryness in water-bath, and residue adds methanol 0.5-2ml makes dissolving, as need testing solution; Other gets ginsenoside Rg1's reference substance and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 chloroform: lower floor's solution of methanol: water=5-50: 2-20: 0.5-10 is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to speckle colour developing at 105 ℃, puts under the 200-500nm ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle 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, medicinal residues add methanol 10-40ml, reflux 10-50 minute, put cold, filter, filtrate evaporate to dryness, residue add methanol 0.5-2ml dissolving, as need testing solution; Other gets Herba Portulacae Grandiflorae control medicinal material 0.5-2g, shines medical material solution in pairs with legal system; According to Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: Ethyl formate: formic acid=2-20: 1-9: 1-9 is developing solvent, launch, take out, dry; Put under the 200-500nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color;
(3) get capsule, tablet or granule 1-5g, porphyrize adds 50% methanol 10-50ml, reflux 0.5-2 hour, put coldly, filter, the filtrate evaporate to dryness, residue adds 50% methanol and dissolves in right amount, is added on 4g, 100～200 purpose neutral alumina posts, with 40% methanol 20-80ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5-2ml makes dissolving, as need testing solution; Other gets the loganin reference substance, adds methanol and makes the solution that every 1ml contains 0.5-2mg, in contrast product solution; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: acetone: dehydrated alcohol: formic acid=1-9: 5-50: 0.5-10: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is heated to clear spot with 10% ethanol solution of sulfuric acid at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle 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 dehydrated alcohol 0.5-2ml dissolving, as need testing solution; Other gets Fructus Ligustri Lucidi control medicinal material 0.2-2g, shines medical material solution in pairs with legal system; Test according to the Chinese Pharmacopoeia thin layer 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 adhesive with the sodium carboxymethyl cellulose, with toluene: ethyl acetate: formic acid=10-50: 1-9: 0.1-0.9 is developing solvent, launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle 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 an immobile phase with 5% phenyl-methylsiloxane; Temperature programming: 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%;
Other capsules of the present invention, tablet or granule should meet Chinese Pharmacopoeia about the pertinent 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 mobile 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 peak should be not less than 3000; Precision takes by weighing the astragaloside reference substance, adds methanol 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, extracting solution evaporate to dryness, residue added and are transferred in the separatory 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, with water saturated n-butanol extraction 3-5 time, 20-60ml at every turn; Merge n-butanol extracting liquid,, discard ammoniacal liquor with ammonia solution 20-60ml washing 1-2 time, n-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 eluting discards water liquid, reuse 70% ethanol 60-100ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter, promptly get need testing solution with 0.45 μ m microporous filter membrane; 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, must not be less than 0.08mg/g in astragaloside C41H68O14; Contain the Radix Astragali in the tablet in astragaloside C41H68O14, must not be less than 0.08mg/g; Contain the Radix Astragali in the granule in astragaloside C41H68O14, 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-1750% methyl-50% phenyl polysiloxane fixative 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, sucking 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, containing cantharidin C10H12O4 in the capsule is 0.025-0.180mg/g; Containing cantharidin C10H12O4 in the tablet is 0.025-0.180mg/g; Containing cantharidin C10H12O4 in the granule is 0.003-0.018mg/g.
11. the method for quality control according to claim 8,9 or 10 described compound cantharidin oral preparations 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.
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