CN1823887A - Quality control method of injection preparation used for treating tumour - Google Patents
Quality control method of injection preparation used for treating tumour Download PDFInfo
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- CN1823887A CN1823887A CNA2005102008812A CN200510200881A CN1823887A CN 1823887 A CN1823887 A CN 1823887A CN A2005102008812 A CNA2005102008812 A CN A2005102008812A CN 200510200881 A CN200510200881 A CN 200510200881A CN 1823887 A CN1823887 A CN 1823887A
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Abstract
A quality control method for the injection to treat tumor includes such steps as checking its characteristics, discriminating by color development reaction on alpha-naphthalenol and the thin-layer identification by referring to ginsenoside Re, Rg1 and Rb and astragaloside A, and measuring the contents of ginsenoside Re and cantharidin.
Description
Technical field: the present invention relates to a kind of method of quality control for the treatment of the ejection preparation of tumor, belong to the technical field of medicine being carried out quality control.
Background technology: the human existing history more than 3000 of tumor of finding.The malignant tumor of epithelium is called cancer, accounts for more than 90% of all malignant tumor.Cancer is a big class disease of serious threat human health, and the patient is in continuous rising.A kind of medicine for the treatment of tumor by the applicant's development, it mainly is prepared from by Mylabris, Radix Ginseng, the Radix Astragali, Radix Et Caulis Acanthopanacis Senticosi, this product has heat-clearing and toxic substances removing, the effect of repercussive eliminating stagnation, at treatment primary hepatocarcinoma, pulmonary carcinoma, rectal cancer, malignant lymphoma, disease aspects such as gynecologic malignant tumor obtain good effect.But how effectively controlling the quality of this product, guarantee the clinical efficacy of this product, is the problem that the applicant studies for a long time always.
Summary of the invention:
The objective of the invention is to: a kind of method of quality control for the treatment of the ejection preparation of tumor is provided, this ejection preparation comprises injection, lyophilized powder and infusion solutions, the present invention studies and screens the assay of main effective ingredient ginsenoside Re and cantharidin in the preparation and the thin layer discriminating of the Radix Ginseng and the Radix Astragali, the method of quality control of being formulated can be controlled the quality of product fully and effectively, thereby has guaranteed the clinical efficacy of said preparation.
The ejection preparation of treatment tumor of the present invention is to constitute like this: calculate according to composition by weight: it mainly is prepared from by Mylabris 0.5-5, Radix Ginseng 20-80, Radix Astragali 50-150, Radix Et Caulis Acanthopanacis Senticosi 100-200.
Ejection preparation of the present invention prepares like this: the Radix Ginseng section, extract 1-5 time with the 20-85% alcohol heating reflux, and merge extractive liquid, filters, and reclaims ethanol, and medicinal liquid is standby; Three flavors such as medicinal residues and all the other Mylabris decoct with water 1-6 time, collecting decoction, filter, filtrate and Radix Ginseng extractive solution merge, with stone sulfur method precipitation process 1-3 time, the gained supernatant adds ethanol to be made and contains the alcohol amount and reach 50-99%, standing over night, get the supernatant decompression recycling ethanol to there not being the alcohol flavor, make injection, infusion solutions or freeze-dried powder preparation respectively according to conventional method again.
Described method of quality control mainly comprise in character, inspection, discriminating, the assay project partly or entirely; Wherein differentiate and comprise with the chromogenic reaction of alpha-Naphthol, with ginsenoside Re, Rg
1, Rb and astragaloside reference substance differentiate for the thin layer of contrast; Assay comprises the assay to ginsenoside Re in the preparation and cantharidin.
The discrimination method of the Radix Ginseng and the Radix Astragali is with ginsenoside Re, Rg in this preparation
1, Rb and astragaloside reference substance be contrast, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ are the thin layer discrimination method of developing solvent.
Discrimination method comprises the part or all of of following project:
(1) getting lyophilized powder respectively adds injection water dissolving or gets infusion solutions and concentrate or directly get injection; Add ethanol and stir evenly, filter, precipitate washing with alcohol 1-5 time, with the precipitate dissolved in distilled water, get 0.5-3ml and put in the test tube, add 2-10% alpha-Naphthol alcoholic solution after the slight fever, shake up, slowly add concentrated sulphuric acid along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder respectively adds injection water dissolving or gets infusion solutions and concentrate or directly get injection; Put in the separatory funnel, with water saturated n-butanol extraction 1-5 time, merge extractive liquid, is put evaporate to dryness in the water-bath, the residue dissolved in distilled water, add on the DA-201 resin column for preparing in advance, wash reuse 20-85% methanol solution eluting with water, collect eluent, evaporate to dryness, residue add methanol makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg
1, Rb and astragaloside reference substance, add dissolve with methanol, in contrast product solution; According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, launches, take out, dry, spray is heated with 10% ethanol solution of sulfuric acid, put under the ultra-violet lamp 200-500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Discrimination method comprises the part or all of of following project more specifically:
(1) getting lyophilized powder 30-100g adds injection water 5-20ml and makes dissolving or get infusion solutions 30-100ml and be concentrated into 5-20ml; Or get injection 5-20ml; Get above-mentioned medicine respectively, add ethanol 20-100ml, stir evenly, filter, precipitate washing with alcohol 1-5 time, precipitate is dissolved with distilled water 2-10ml, get 0.5-3ml and put in the test tube, add 2-10% alpha-Naphthol alcoholic solution 2-10 after the slight fever and drip, shake up, slowly add concentrated sulphuric acid 0.2-2ml along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder 200-400g adds injection water 40-60ml and makes dissolving or get infusion solutions 200-400ml and be concentrated into 40-60ml; Or get injection 40-60ml; Get above-mentioned medicine respectively, put in the separatory funnel, with water saturated n-butanol extraction 1-5 time, merge extractive liquid,, put evaporate to dryness in the water-bath, residue dissolves with distilled water 1-10ml, behind the DA-201 resin of internal diameter 1～1.5cm, the long 15cm that adding prepares in advance, filling 12cm, recharge on the DA-201 resin column of 2g neutral alumina water 50-150ml washing, reuse 20-85% methanol solution 30-100ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5-3ml makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg
1, Rb and astragaloside reference substance, add methanol and make the mixed solution that every 1ml contains 0.5-3mg, in contrast product solution; According to the thin layer chromatography test, draw each 3-20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ of heating, put under the ultra-violet lamp 200-500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Ginsenoside Re's content assaying method is to be the spectrophotography of contrast with ginsenoside Re's reference substance in this preparation.
The content assaying method of cantharidin is to be contrast with the cantharidin reference substance in this preparation, is the gas chromatography of fixative with carbowax-20M and methyl silicone rubber SE-30.
Content assaying method comprises:
(1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, puts respectively in the 10ml tool plug test tube, puts the water-bath Back stroke and desolvates, take out immediately, put coldly, add 2-10% vanillin glacial acetic acid solution and perchloric acid, shake up, put in the water-bath and heat, take out, use water cooling, add glacial acetic acid, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder respectively and add injection water dissolving or get infusion solutions and concentrate or directly get injection; Put in the separatory funnel, use chloroform extraction 1-5 time, the combined chloroform extracting solution, with distilled water wash 1-5 time, discard chloroform solution, washing liquid and above-mentioned water layer merge, put in the separatory funnel,, merge n-butanol extracting liquid with water saturated n-butanol extraction 1-6 time, add anhydrous sodium sulfate, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, evaporate to dryness, use the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, treat that liquid level is near cotton layer after, use the distilled water eluting, discard washing liquid, use the 40-90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and adds the chloroform dissolving, shakes up, in contrast product solution;
The preparation of need testing solution: get lyophilized powder respectively and add injection water dissolving or get infusion solutions and concentrate or directly get injection; Put in the tool plug test tube, add the 1-3mol/L sulfuric acid solution, shake up, the accurate again chloroform that adds, close plug, jolting, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
Content assaying method comprises more specifically:
(1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol and makes and contain ginsenoside Re 1-2mg among every 1ml, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, put respectively in the 10ml tool plug test tube, putting the water-bath Back stroke desolvates, take out immediately, put cold, add 2-10% vanillin glacial acetic acid solution 0.1-1ml, perchloric acid 0.5-1.5ml, shake up, put in the 40-80 ℃ of water-bath and heated 10-30 minute, take out, with water cooling 0.5-5 minute, add glacial acetic acid 2-10ml, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap at 300-700nm wavelength place, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder 60-150g and add injection water 10-30ml and make dissolving or get infusion solutions 50-200ml and be concentrated into 10-30ml; Or get injection 10-30ml; Precision is measured above-mentioned medicine respectively, puts in the separatory funnel, uses chloroform extraction 1-5 time, the combined chloroform extracting solution is used distilled water wash 1-5 time, discards chloroform solution, washing liquid and above-mentioned water layer merge, and put in the separatory funnel, with water saturated n-butanol extraction 1-6 time, merge n-butanol extracting liquid, add anhydrous sodium sulfate 1-10g, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, and evaporate to dryness is used the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, after treating that liquid level is near cotton layer,, discard washing liquid with distilled water 100ml eluting, use the 40-90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 0.05-0.1mg, in contrast product solution;
The preparation of need testing solution: get lyophilized powder 30-100g and add injection water 5-20ml and make dissolving or get infusion solutions 30-100ml and be concentrated into 5-20ml; Or get injection 5-20ml; Get above-mentioned medicine respectively, put in the tool plug test tube, add 1-3mol/L sulfuric acid solution 0.5-3ml, shake up, the accurate again 0.5-3ml chloroform that adds, close plug, jolting 1-10 minute, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and each 2-15 μ l of need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
Described method of quality control comprises:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Differentiate: (1) is got lyophilized powder respectively and is added the dissolving of injection water or get infusion solutions and concentrate or directly get injection; Add ethanol and stir evenly, filter, precipitate washing with alcohol 1-5 time, with the precipitate dissolved in distilled water, get 0.5-3ml and put in the test tube, add 2-10% alpha-Naphthol alcoholic solution after the slight fever, shake up, slowly add concentrated sulphuric acid along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder respectively adds injection water dissolving or gets infusion solutions and concentrate or directly get injection; Put in the separatory funnel, with water saturated n-butanol extraction 1-5 time, merge extractive liquid, is put evaporate to dryness in the water-bath, the residue dissolved in distilled water, add on the DA-201 resin column for preparing in advance, wash reuse 20-85% methanol solution eluting with water, collect eluent, evaporate to dryness, residue add methanol makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg
1, Rb and astragaloside reference substance, add dissolve with methanol, in contrast product solution; According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, launches, take out, dry, spray is heated with 10% ethanol solution of sulfuric acid, put under the ultra-violet lamp 200-500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
Check:
PH value: should be 3.0-6.0;
Heavy metal: get lyophilized powder 40-80g and add injection water 10ml and make dissolving or get infusion solutions 2ml or get injection 2ml, put in the crucible, evaporate to dryness in the water-bath is checked according to Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
Pyrogen: get lyophilized powder 100-200g respectively and add injection water 30ml and make dissolving or get infusion solutions 30ml or get injection 30ml; Be diluted to 80-150ml with sodium chloride injection or 5～10% glucose injections,, check according to the Chinese Pharmacopoeia pyrogen test by the dosage of rabbit body weight 1kg injection 10ml, should be up to specification;
Other: injection of the present invention, lyophilized powder or infusion solutions should meet Chinese Pharmacopoeia about the pertinent regulations under the injection item;
Assay:
(1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, puts respectively in the 10ml tool plug test tube, puts the water-bath Back stroke and desolvates, take out immediately, put coldly, add 2-10% vanillin glacial acetic acid solution and perchloric acid, shake up, put in the water-bath and heat, take out, use water cooling, add glacial acetic acid, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder respectively and add injection water dissolving or get infusion solutions and concentrate or directly get injection; Put in the separatory funnel, use chloroform extraction 1-5 time, the combined chloroform extracting solution, with distilled water wash 1-5 time, discard chloroform solution, washing liquid and above-mentioned water layer merge, put in the separatory funnel,, merge n-butanol extracting liquid with water saturated n-butanol extraction 1-6 time, add anhydrous sodium sulfate, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, evaporate to dryness, use the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, treat that liquid level is near cotton layer after, use the distilled water eluting, discard washing liquid, use the 40-90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and adds the chloroform dissolving, shakes up, in contrast product solution;
The preparation of need testing solution: get lyophilized powder respectively and add injection water dissolving or get infusion solutions and concentrate or directly get injection; Put in the tool plug test tube, add the 1-3mol/L sulfuric acid solution, shake up, the accurate again chloroform that adds, close plug, jolting, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
Find after deliberation, adopt the quality of following method of quality control, be more conducive to guarantee the clinical efficacy of this preparation easier control preparation of the present invention.So described method of quality control also can comprise:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Differentiate: (1) is got lyophilized powder 30-100g and is added injection water 5-20ml and make dissolving or get infusion solutions 30-100ml and be concentrated into 5-20ml; Or get injection 5-20ml; Get above-mentioned medicine respectively, add ethanol 20-100ml, stir evenly, filter, precipitate washing with alcohol 1-5 time, precipitate is dissolved with distilled water 2-10ml, get 0.5-3ml and put in the test tube, add 2-10% alpha-Naphthol alcoholic solution 2-10 after the slight fever and drip, shake up, slowly add concentrated sulphuric acid 0.2-2ml along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder 200-400g adds injection water 40-60ml and makes dissolving or get infusion solutions 200-400ml and be concentrated into 40-60ml; Or get injection 40-60ml; Get above-mentioned medicine respectively, put in the separatory funnel, with water saturated n-butanol extraction 1-5 time, merge extractive liquid,, put evaporate to dryness in the water-bath, residue dissolves with distilled water 1-10ml, behind the DA-201 resin of internal diameter 1～1.5cm, the long 15cm that adding prepares in advance, filling 12cm, recharge on the DA-201 resin column of 2g neutral alumina water 50-150ml washing, reuse 20-85% methanol solution 30-100ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5-3ml makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg
1, Rb and astragaloside reference substance, add methanol and make the mixed solution that every 1ml contains 0.5-3mg, in contrast product solution; According to the thin layer chromatography test, draw each 3-20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ of heating, put under the ultra-violet lamp 200-500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
Check:
PH value: should be 3.0-6.0;
Heavy metal: get lyophilized powder 40-80g and add injection water 10ml and make dissolving or get infusion solutions 2ml or get injection 2ml, put in the crucible, evaporate to dryness in the water-bath is checked according to Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
Pyrogen: get lyophilized powder 100-200g respectively and add injection water 30ml and make dissolving or get infusion solutions 30ml or get injection 30ml; Be diluted to 80-150ml with sodium chloride injection or 5～10% glucose injections,, check according to the Chinese Pharmacopoeia pyrogen test by the dosage of rabbit body weight 1kg injection 10ml, should be up to specification;
Other: injection of the present invention, lyophilized powder or infusion solutions should meet Chinese Pharmacopoeia about the pertinent regulations under the injection item;
Assay:
(1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol and makes and contain ginsenoside Re 1-2mg among every 1ml, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, put respectively in the 10ml tool plug test tube, putting the water-bath Back stroke desolvates, take out immediately, put cold, add 2-10% vanillin glacial acetic acid solution 0.1-1ml, perchloric acid 0.5-1.5ml, shake up, put in the 40-80 ℃ of water-bath and heated 10-30 minute, take out, with water cooling 0.5-5 minute, add glacial acetic acid 2-10ml, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap at 300-700nm wavelength place, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder 60-150g and add injection water 10-30ml and make dissolving or get infusion solutions 50-200ml and be concentrated into 10-30ml; Or get injection 10-30ml; Precision is measured above-mentioned medicine respectively, puts in the separatory funnel, uses chloroform extraction 1-5 time, the combined chloroform extracting solution is used distilled water wash 1-5 time, discards chloroform solution, washing liquid and above-mentioned water layer merge, and put in the separatory funnel, with water saturated n-butanol extraction 1-6 time, merge n-butanol extracting liquid, add anhydrous sodium sulfate 1-10g, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, and evaporate to dryness is used the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, after treating that liquid level is near cotton layer,, discard washing liquid with distilled water 100ml eluting, use the 40-90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 0.05-0.1mg, in contrast product solution;
The preparation of need testing solution: get lyophilized powder 30-100g and add injection water 5-20ml and make dissolving or get infusion solutions 30-100ml and be concentrated into 5-20ml; Or get injection 5-20ml; Get above-mentioned medicine respectively, put in the tool plug test tube, add 1-3mol/L sulfuric acid solution 0.5-3ml, shake up, the accurate again 0.5-3ml chloroform that adds, close plug, jolting 1-10 minute, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and each 2-15 μ l of need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
In this preparation, Radix Ginseng has the effect of enhancing immunity, and Mylabris has antineoplastic action.Radix Ginseng and Mylabris are the medicine that plays a major role in this product, and ginsenoside Re and cantharidin are respectively the main effective ingredient in Radix Ginseng and the Mylabris, so the present invention tackles the ginsenoside Re and cantharidin carries out assay, can effectively control the quality of this product, but how effectively measuring the content of ginsenoside Re and cantharidin in the said preparation, is difficult point of the present invention place.Through research, we find, adopt spectrophotography, and employing chloroform extraction medicine, and then use water saturated n-butanol extraction, the method for crossing macroporous adsorbent resin behind the adding anhydrous sodium sulfate prepares need testing solution, can effectively measure the content of ginsenoside Re in the said preparation; Adopt gas chromatography, chromatographic condition and system suitability test are: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Can effectively measure the content of cantharidin in the preparation with this method.In the method for preparing the cantharidin need testing solution, we do not use the K-D concentrator, have reduced inspection cost in addition.
The present invention has also carried out the thin layer Study on Identification to the Radix Ginseng and the Radix Astragali, and with ginsenoside Re, Rg
1, Rb and astragaloside reference substance be for contrast, adopts water saturated n-butanol extraction, cross the method for resin column then and extract medicine, the preparation need testing solution; With chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, with Radix Ginseng and the Milkvetch Root in this method discriminating preparation, its separating degree is good, the speckle colour developing is clear, and negative control is noiseless, the method favorable reproducibility.So can effectively control the quality of ejection preparation of treatment tumor with the inventive method, guarantee the clinical efficacy of said preparation.
Method of quality control of the present invention is the preferred plan that obtains through a large amount of screenings, and following experimentation is a preferred process of the present invention.
One, ginsenoside Re's content assaying method research
1, need testing solution preparation method research:
Method 1: the thing of getting it filled, put in the separatory funnel, use chloroform extraction, the combined chloroform extracting solution is used distilled water wash, discards chloroform solution, washing liquid and above-mentioned water layer merge, and put in the separatory funnel, use water saturated n-butanol extraction, merge n-butanol extracting liquid, add anhydrous sodium sulfate, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, evaporate to dryness, use the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, treat that liquid level is near cotton layer after, use the distilled water eluting, discard washing liquid, use 70% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, shake up, promptly.
Method 2: the thing of getting it filled, put in the evaporating dish, in water-bath, concentrate, add the chloroform jolting and extract, discard chloroform, add water saturated n-butyl alcohol jolting again and extract 3 times, merge n-butyl alcohol, add ammonia solution and wash 1 time, discard alkali liquor, butanol solution is put evaporate to dryness in the water-bath, and residue adds dissolve with methanol, promptly.
| Sample | Method 1 (mg/ml) | Method 2 (mg/ml) |
| 1 | 0.254 | 0.231 |
| 2 | 0.243 | 0.229 |
| 3 | 0.261 | 0.235 |
According to result of the test as can be known, employing method 1 preparation need testing solution, the ginsenoside Re extracts more complete.
Two, cantharidin content assaying method research
1, need testing solution preparation method research:
Method 1: the thing of getting it filled, put in the tool plug test tube, add the 1.8mol/L sulfuric acid solution, shake up, add chloroform again, close plug, jolting 3 minutes, standing demix, centrifugal in case of necessity, get subnatant promptly.
Method 2: the thing of getting it filled, add the 1.8mol/L sulfuric acid solution, use chloroform extraction 3 times, combined chloroform liquid concentrates and standardize solution with the K-D concentrator, promptly.
| Sample | Method 1 (mg/ml) | Method 2 (mg/ml) |
| 1 | 0.00151 | 0.00154 |
| 2 | 0.00175 | 0.00172 |
| 3 | 0.00208 | 0.00212 |
According to experimental result as can be known, the cantharidin content that extracts of two kinds of methods does not have significant difference.But, increased inspection cost owing to used the K-D concentrator in the method 2.Understand according to another the applicant, because the K-D concentrator is used for the detection of food more, that uses in medicine detects is few, and domestic most drug K-D concentrator that check does not all have, so employing method 1 detects the content of cantharidin in this product among the present invention, not only can reduce inspection cost relatively, also make things convenient for each province medicine inspecting institute that this product is detected.
Three, Radix Ginseng, Radix Astragali thin layer Study on Identification
Need testing solution preparation method one: the thing of getting it filled, put in the separatory funnel, with water saturated n-butanol extraction 2 times, merge extractive liquid,, put evaporate to dryness in the water-bath, the residue dissolved in distilled water adds on the DA-201 resin column for preparing in advance, wash with water, reuse 40% methanol solution eluting is collected eluent, evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Be equipped with shortage of staff's ginseng, Radix Astragali negative controls with legal system.
Need testing solution preparation method two: the thing of getting it filled, in the filtration paper cylinder of packing into, put in the apparatus,Soxhlet's, adding diethyl ether, it is colourless to be extracted into, and discards ether solution, adds to add methanol extraction after the methanol soaked overnight again to colourless, use the small amount of methanol washing container, merge extractive liquid, and cleaning mixture, water bath method, the fusion of residue water slight fever, with the saturated n-butanol extraction of 0.1mol/L sodium hydroxide 6 times, merge n-butanol extracting liquid, water bath method, residue adds dissolve with methanol; Be equipped with shortage of staff's ginseng, Radix Astragali negative controls with legal system.
Developing solvent is selected: respectively with the mixed solution of chloroform, ethyl acetate, acetonitrile, water different proportion; The mixed solution of chloroform, ethyl acetate, methanol, water different proportion; The mixed solution of chloroform, methanol, water different proportion is developing solvent.
The result: employing method one preparation need testing solution, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, its separating degree is good, the speckle colour developing is clear, and negative control is noiseless, the method favorable reproducibility.Best developing solvent is: chloroform: ethyl acetate: methanol: water=4: 8: 3: 4, lower floor's solution of placing below 10 ℃.
Compared with prior art, the present invention studies and screens the assay of main effective ingredient ginsenoside Re and cantharidin in the preparation and the thin layer discriminating of the Radix Ginseng and the Radix Astragali, the method of quality control that is adopted is scientific and reasonable, the accuracy height, favorable reproducibility, can not only control the quality of the ejection preparation of treatment tumor fully and effectively, guarantee the clinical efficacy of said preparation, and the method that is adopted reduce inspection cost relatively.
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:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Differentiate: (1) is got lyophilized powder 60g and is added injection water 10ml and make dissolving or get infusion solutions 60ml and be concentrated into 10ml; Or get injection 10ml; Get above-mentioned medicine respectively, add ethanol 50ml, stir evenly, filter, precipitate washing with alcohol 2 times, precipitate is dissolved with distilled water 5ml, get 1ml and put in the test tube, add 50 of 5% alpha-Naphthol alcoholic solution after the slight fever, shake up, slowly add concentrated sulphuric acid 0.5ml along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder 300g adds injection water 50ml and makes dissolving or get infusion solutions 300ml and be concentrated into 30ml; Or get injection 50ml; Get above-mentioned medicine respectively, put in the separatory funnel, with water saturated n-butanol extraction 2 times, each 30ml, merge extractive liquid, is put evaporate to dryness in the water-bath, residue adds the DA-201 resin column (internal diameter 1～1.5cm, the long 15cm that prepare in advance with distilled water 3ml dissolving, after filling the DA-201 resin of 12cm, recharge the 2g neutral alumina) on, water 100ml washing, reuse 40% methanol solution 50ml eluting is collected eluent, evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg
1, Rb and astragaloside reference substance, add methanol and make the mixed solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol: water=4: 8: 3: lower floor's solution of placing below 4,10 ℃ is developing solvent, launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ of heating several minutes, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
Check:
PH value: should be 3.8-5.0;
Heavy metal: get lyophilized powder 20g and add injection water 2ml and make dissolving or get infusion solutions 2ml or get injection 2ml, put in the crucible, evaporate to dryness in the water-bath is checked according to Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
Pyrogen: get lyophilized powder 150g respectively and add injection water 30ml and make dissolving or get infusion solutions 30ml or get injection 30ml; Be diluted to 100ml with sodium chloride injection or 5～10% glucose injections,, check according to the Chinese Pharmacopoeia pyrogen test by the dosage of rabbit body weight 1kg injection 10ml, should be up to specification;
Other: injection of the present invention, lyophilized powder or infusion solutions should meet Chinese Pharmacopoeia about the pertinent regulations under the injection item;
Assay: (1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol and makes and contain ginsenoside Re 1.6mg among every 1ml, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, put respectively in the 10ml tool plug test tube, putting the water-bath Back stroke desolvates, take out immediately, put cold, add 5% vanillin glacial acetic acid solution 0.2ml, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, with water cooling 2 minutes, add glacial acetic acid 5ml, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap at 544nm wavelength place, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder 100g and add injection water 20ml and make dissolving or get infusion solutions 120ml and be concentrated into 20ml; Or get injection 20ml; Precision is measured above-mentioned medicine respectively, puts in the separatory funnel, uses chloroform extraction 3 times, each 20ml, the combined chloroform extracting solution is used distilled water wash 2 times, each 5ml discards chloroform solution, and washing liquid and above-mentioned water layer merge, put in the separatory funnel, with water saturated n-butanol extraction 3 times, each 50ml, merge n-butanol extracting liquid, add anhydrous sodium sulfate 3g, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, evaporate to dryness is used the dissolved in distilled water residue, is added on the macroporous adsorbent resin DA-201 post, after treating that liquid level is near cotton layer, with distilled water 100ml eluting, the control flow velocity is 0.4ml/min, discards washing liquid, with 70% ethanol 80ml eluting, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates.In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: 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; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 0.01mg, in contrast product solution;
The preparation of need testing solution: get lyophilized powder 60g and add injection water 10ml and make dissolving or get infusion solutions 60ml and be concentrated into 10ml; Or get injection 10ml; Get above-mentioned medicine respectively, put in the tool plug test tube, add 1.8mol/L sulfuric acid solution 1ml, shake up, the accurate again 1ml chloroform that adds, close plug, jolting 3 minutes, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and each 4 μ l of need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
Embodiments of the invention 2:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Differentiate: get lyophilized powder 30g and add injection water 5ml and make dissolving or get infusion solutions 30ml and be concentrated into 5ml; Or get injection 5ml; Get above-mentioned medicine respectively, add ethanol 20ml, stir evenly, filter, precipitate washing with alcohol 1 time, precipitate is dissolved with distilled water 2ml, get 0.5ml and put in the test tube, add 2 of 2% alpha-Naphthol alcoholic solution after the slight fever, shake up, slowly add concentrated sulphuric acid 0.2ml along tube wall again, at two liquid junction place displaing amaranth rings;
Check:
PH value: should be 3.0-5.0;
Pyrogen: get lyophilized powder 100g respectively and add injection water 30ml and make dissolving or get infusion solutions 30ml or get injection 30ml; Be diluted to 80ml with sodium chloride injection or 5～10% glucose injections,, check according to the Chinese Pharmacopoeia pyrogen test by the dosage of rabbit body weight 1kg injection 10ml, should be up to specification;
Assay: cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5% and 2%, 1: 9 mix the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 0.05mg, in contrast product solution;
The preparation of need testing solution: get lyophilized powder 30g and add injection water 5ml and make dissolving or get infusion solutions 30ml and be concentrated into 5ml; Or get injection 5ml; Get above-mentioned medicine respectively, put in the tool plug test tube, add 1mol/L sulfuric acid solution 0.5ml, shake up, the accurate again 0.5ml chloroform that adds, close plug, jolting 1 minute, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and each 2 μ l of need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
Embodiments of the invention 3:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Differentiate: get lyophilized powder 200g and add injection water 40ml and make dissolving or get infusion solutions 200ml and be concentrated into 40ml; Or get injection 40ml; Get above-mentioned medicine respectively, put in the separatory funnel, with water saturated n-butanol extraction 1 time, merge extractive liquid, is put evaporate to dryness in the water-bath, and residue dissolves with distilled water 1ml, the DA-201 resin column that adding prepares in advance (internal diameter 1～1.5cm, long 15cm is behind the DA-201 resin of filling 12cm, recharge the 2g neutral alumina) on, water 50ml washing, reuse 20% methanol solution 30ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5ml makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg
1, Rb and astragaloside reference substance, add methanol and make the mixed solution that every 1ml contains 0.5mg, in contrast product solution; According to the thin layer chromatography test, draw each 3 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=10: 5: 0.5: lower floor's solution of placing below 10,10 ℃ is developing solvent, launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ of heating, put under the ultra-violet lamp 200nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
Assay: ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol and makes and contain ginsenoside Re 1mg among every 1ml, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, put respectively in the 10ml tool plug test tube, putting the water-bath Back stroke desolvates, take out immediately, put cold, add 2% vanillin glacial acetic acid solution 0.1ml, perchloric acid 0.5ml, shake up, put in 40 ℃ of water-baths and heated 10 minutes, take out, with water cooling 0.5 minute, add glacial acetic acid 2ml, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap at 300nm wavelength place, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder 60g and add injection water 10ml and make dissolving or get infusion solutions 50ml and be concentrated into 10ml; Or get injection 10ml; Precision is measured above-mentioned medicine respectively, puts in the separatory funnel, uses chloroform extraction 1 time, the combined chloroform extracting solution is used distilled water wash 1 time, discards chloroform solution, washing liquid and above-mentioned water layer merge, and put in the separatory funnel, with water saturated n-butanol extraction 1 time, merge n-butanol extracting liquid, add anhydrous sodium sulfate 1g, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, and evaporate to dryness is used the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, treat that liquid level is near cotton layer after, with distilled water 100ml eluting, the control flow velocity is 0.4ml/min, discard washing liquid, use 40% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml.
Embodiments of the invention 4:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Check:
Heavy metal: get lyophilized powder 40g and add injection water 10ml and make dissolving or get infusion solutions 2ml or get injection 2ml, put in the crucible, evaporate to dryness in the water-bath is checked according to Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
Other: injection of the present invention, lyophilized powder or infusion solutions should meet Chinese Pharmacopoeia about the pertinent regulations under the injection item;
Assay: (1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol and makes and contain ginsenoside Re 2mg among every 1ml, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, put respectively in the 10ml tool plug test tube, putting the water-bath Back stroke desolvates, take out immediately, put cold, add 10% vanillin glacial acetic acid solution 1ml, perchloric acid 1.5ml, shake up, put in 80 ℃ of water-baths and heated 30 minutes, take out, with water cooling 5 minutes, add glacial acetic acid 10ml, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap at 700nm wavelength place, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder 150g and add injection water 30ml and make dissolving or get infusion solutions 200ml and be concentrated into 30ml; Or get injection 30ml; Precision is measured above-mentioned medicine respectively, puts in the separatory funnel, uses chloroform extraction 5 times, each 10ml, the combined chloroform extracting solution is used distilled water wash 5 times, each 5ml discards chloroform solution, and washing liquid and above-mentioned water layer merge, put in the separatory funnel, with water saturated n-butanol extraction 6 times, each 30ml, merge n-butanol extracting liquid, add anhydrous sodium sulfate 10g, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, evaporate to dryness is used the dissolved in distilled water residue, is added on the macroporous adsorbent resin DA-201 post, after treating that liquid level is near cotton layer, with distilled water 100ml eluting, the control flow velocity is 0.4ml/min, discards washing liquid, use 90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 20% and 10%, 9: 1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 0.1mg, in contrast product solution;
The preparation of need testing solution: get lyophilized powder 100g and add injection water 20ml and make dissolving or get infusion solutions 100ml and be concentrated into 20ml; Or get injection 20ml; Get above-mentioned medicine respectively, put in the tool plug test tube, add 3mol/L sulfuric acid solution 3ml, shake up, the accurate again 3ml chloroform that adds, close plug, jolting 10 minutes, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and each 15 μ l of need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
Embodiments of the invention 5:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Differentiate: (1) is got lyophilized powder 100g and is added injection water 20ml and make dissolving or get infusion solutions 100ml and be concentrated into 20ml; Or get injection 20ml; Get above-mentioned medicine respectively, add ethanol 100ml, stir evenly, filter, precipitate washing with alcohol 5 times, precipitate is dissolved with distilled water 10ml, get 3ml and put in the test tube, add 10 of 10% alpha-Naphthol alcoholic solution after the slight fever, shake up, slowly add concentrated sulphuric acid 2ml along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder 400g adds injection water 60ml and makes dissolving or get infusion solutions 400ml and be concentrated into 60ml; Or get injection 60ml; Get above-mentioned medicine respectively, put in the separatory funnel, with water saturated n-butanol extraction 5 times, each 15ml, merge extractive liquid, is put evaporate to dryness in the water-bath, residue adds the DA-201 resin column (internal diameter 1～1.5cm, the long 15cm that prepare in advance with distilled water 10ml dissolving, after filling the DA-201 resin of 12cm, recharge the 2g neutral alumina) on, water 150ml washing, reuse 85% methanol solution 100ml eluting is collected eluent, evaporate to dryness, residue adds methanol 3ml makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg
1, Rb and astragaloside reference substance, add methanol and make the mixed solution that every 1ml contains 3mg, in contrast product solution; According to the thin layer chromatography test, draw each 20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1: 15: 6: lower floor's solution of placing below 1,10 ℃ is developing solvent, launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ of heating, put under the ultra-violet lamp 500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
Check:
PH value: should be 3.8-6.0;
Heavy metal: get lyophilized powder 80g and add injection water 10ml and make dissolving or get infusion solutions 2ml or get injection 2ml, put in the crucible, evaporate to dryness in the water-bath is checked according to Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
Pyrogen: get lyophilized powder 200g respectively and add injection water 30ml and make dissolving or get infusion solutions 30ml or get injection 30ml; Be diluted to 150ml with sodium chloride injection or 5～10% glucose injections,, check according to the Chinese Pharmacopoeia pyrogen test by the dosage of rabbit body weight 1kg injection 10ml, should be up to specification;
Other: injection of the present invention, lyophilized powder or infusion solutions should meet Chinese Pharmacopoeia about the pertinent regulations under the injection item.
Claims (10)
1. method of quality control for the treatment of the ejection preparation of tumor, this ejection preparation comprises injection, lyophilized powder and infusion solutions, it is characterized in that: described method of quality control mainly comprise in character, inspection, discriminating, the assay project partly or entirely; Wherein differentiate and comprise that with the chromogenic reaction of alpha-Naphthol, with ginsenoside Re, Rg1, Rb and astragaloside reference substance be the thin layer discriminating of contrast; Assay comprises the assay to ginsenoside Re in the preparation and cantharidin.
2. according to the method for quality control of the ejection preparation of the described treatment tumor 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 Re, Rg1, Rb and astragaloside reference substance in this preparation, and with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ are the thin layer discrimination method of developing solvent.
3. according to the method for quality control of the ejection preparation of claim 1 or 2 described treatment tumors, it is characterized in that: discrimination method comprise following project partly or entirely:
(1) getting lyophilized powder respectively adds injection water dissolving or gets infusion solutions and concentrate or directly get injection; Add ethanol and stir evenly, filter, precipitate washing with alcohol 1-5 time, with the precipitate dissolved in distilled water, get 0.5-3ml and put in the test tube, add 2-10% alpha-Naphthol alcoholic solution after the slight fever, shake up, slowly add concentrated sulphuric acid along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder respectively adds injection water dissolving or gets infusion solutions and concentrate or directly get injection; Put in the separatory funnel, with water saturated n-butanol extraction 1-5 time, merge extractive liquid, is put evaporate to dryness in the water-bath, the residue dissolved in distilled water, add on the DA-201 resin column for preparing in advance, wash reuse 20-85% methanol solution eluting with water, collect eluent, evaporate to dryness, residue add methanol makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg1, Rb and astragaloside reference substance, adds dissolve with methanol, in contrast product solution; According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, launches, take out, dry, spray is heated with 10% ethanol solution of sulfuric acid, put under the ultra-violet lamp 200-500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
4. according to the method for quality control of the ejection preparation of the described treatment tumor of claim 3, it is characterized in that: concrete discrimination method comprise following project partly or entirely:
(1) getting lyophilized powder 30-100g adds injection water 5-20ml and makes dissolving or get infusion solutions 30-100ml and be concentrated into 5-20ml; Or get injection 5-20ml; Get above-mentioned medicine respectively, add ethanol 20-100ml, stir evenly, filter, precipitate washing with alcohol 1-5 time, precipitate is dissolved with distilled water 2-10ml, get 0.5-3ml and put in the test tube, add 2-10% alpha-Naphthol alcoholic solution 2-10 after the slight fever and drip, shake up, slowly add concentrated sulphuric acid 0.2-2ml along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder 200-400g adds injection water 40-60ml and makes dissolving or get infusion solutions 200-400ml and be concentrated into 40-60ml; Or get injection 40-60ml; Get above-mentioned medicine respectively, put in the separatory funnel, with water saturated n-butanol extraction 1-5 time, merge extractive liquid,, put evaporate to dryness in the water-bath, residue dissolves with distilled water 1-10ml, behind the DA-201 resin of internal diameter 1～1.5cm, the long 15cm that adding prepares in advance, filling 12cm, recharge on the DA-201 resin column of 2g neutral alumina water 50-150ml washing, reuse 20-85% methanol solution 30-100ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5-3ml makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg1, Rb and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 0.5-3mg, in contrast product solution; According to the thin layer chromatography test, draw each 3-20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ of heating, put under the ultra-violet lamp 200-500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
5. according to the method for quality control of the ejection preparation of the described treatment tumor of claim 1, it is characterized in that: ginsenoside Re's content assaying method is to be the spectrophotography of contrast with ginsenoside Re's reference substance in this preparation.
6. according to the method for quality control of the ejection preparation of the described treatment tumor of claim 1, it is characterized in that: the content assaying method of cantharidin is to be contrast with the cantharidin reference substance in this preparation, is the gas chromatography of fixative with carbowax-20M and methyl silicone rubber SE-30.
7. according to the method for quality control of the ejection preparation of claim 1,5 or 6 described treatment tumors, it is characterized in that: content assaying method comprises:
(1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, puts respectively in the 10ml tool plug test tube, puts the water-bath Back stroke and desolvates, take out immediately, put coldly, add 2-10% vanillin glacial acetic acid solution and perchloric acid, shake up, put in the water-bath and heat, take out, use water cooling, add glacial acetic acid, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder respectively and add injection water dissolving or get infusion solutions and concentrate or directly get injection; Put in the separatory funnel, use chloroform extraction 1-5 time, the combined chloroform extracting solution, with distilled water wash 1-5 time, discard chloroform solution, washing liquid and above-mentioned water layer merge, put in the separatory funnel,, merge n-butanol extracting liquid with water saturated n-butanol extraction 1-6 time, add anhydrous sodium sulfate, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, evaporate to dryness, use the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, treat that liquid level is near cotton layer after, use the distilled water eluting, discard washing liquid, use the 40-90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and adds the chloroform dissolving, shakes up, in contrast product solution;
The preparation of need testing solution: get lyophilized powder respectively and add injection water dissolving or get infusion solutions and concentrate or directly get injection; Put in the tool plug test tube, add the 1-3mol/L sulfuric acid solution, shake up, the accurate again chloroform that adds, close plug, jolting, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
8. according to the method for quality control of the ejection preparation of the described treatment tumor of claim 7, it is characterized in that: concrete content assaying method comprises:
(1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol and makes and contain ginsenoside Re 1-2mg among every 1ml, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, put respectively in the 10ml tool plug test tube, putting the water-bath Back stroke desolvates, take out immediately, put cold, add 2-10% vanillin glacial acetic acid solution 0.1-1ml, perchloric acid 0.5-1.5ml, shake up, put in the 40-80 ℃ of water-bath and heated 10-30 minute, take out, with water cooling 0.5-5 minute, add glacial acetic acid 2-10ml, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap at 300-700nm wavelength place, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder 60-150g and add injection water 10-30ml and make dissolving or get infusion solutions 50-200ml and be concentrated into 10-30ml; Or get injection 10-30ml; Precision is measured above-mentioned medicine respectively, puts in the separatory funnel, uses chloroform extraction 1-5 time, the combined chloroform extracting solution is used distilled water wash 1-5 time, discards chloroform solution, washing liquid and above-mentioned water layer merge, and put in the separatory funnel, with water saturated n-butanol extraction 1-6 time, merge n-butanol extracting liquid, add anhydrous sodium sulfate 1-10g, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, and evaporate to dryness is used the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, after treating that liquid level is near cotton layer,, discard washing liquid with distilled water 100ml eluting, use the 40-90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 0.05-0.1mg, in contrast product solution;
The preparation of need testing solution: get lyophilized powder 30-100g and add injection water 5-20ml and make dissolving or get infusion solutions 30-100ml and be concentrated into 5-20ml; Or get injection 5-20ml; Get above-mentioned medicine respectively, put in the tool plug test tube, add 1-3mol/L sulfuric acid solution 0.5-3ml, shake up, the accurate again 0.5-3ml chloroform that adds, close plug, jolting 1-10 minute, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and each 2-15 μ l of need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
9. according to the method for quality control of the ejection preparation of the described treatment tumor of claim 1, it is characterized in that: described method of quality control comprises:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Differentiate: (1) is got lyophilized powder respectively and is added the dissolving of injection water or get infusion solutions and concentrate or directly get injection; Add ethanol and stir evenly, filter, precipitate washing with alcohol 1-5 time, with the precipitate dissolved in distilled water, get 0.5-3ml and put in the test tube, add 2-10% alpha-Naphthol alcoholic solution after the slight fever, shake up, slowly add concentrated sulphuric acid along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder respectively adds injection water dissolving or gets infusion solutions and concentrate or directly get injection; Put in the separatory funnel, with water saturated n-butanol extraction 1-5 time, merge extractive liquid, is put evaporate to dryness in the water-bath, the residue dissolved in distilled water, add on the DA-201 resin column for preparing in advance, wash reuse 20-85% methanol solution eluting with water, collect eluent, evaporate to dryness, residue add methanol makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg1, Rb and astragaloside reference substance, adds dissolve with methanol, in contrast product solution; According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, launches, take out, dry, spray is heated with 10% ethanol solution of sulfuric acid, put under the ultra-violet lamp 200-500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
Check:
PH value: should be 3.0-6.0;
Heavy metal: get lyophilized powder 40-80g and add injection water 10ml and make dissolving or get infusion solutions 2ml or get injection 2ml, put in the crucible, evaporate to dryness in the water-bath is checked according to Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
Pyrogen: get lyophilized powder 100-200g respectively and add injection water 30ml and make dissolving or get infusion solutions 30ml or get injection 30ml; Be diluted to 80-150ml with sodium chloride injection or 5～10% glucose injections,, check according to the Chinese Pharmacopoeia pyrogen test by the dosage of rabbit body weight 1kg injection 10ml, should be up to specification;
Other: injection of the present invention, lyophilized powder or infusion solutions should meet Chinese Pharmacopoeia about the pertinent regulations under the injection item;
Assay: (1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, puts respectively in the 10ml tool plug test tube, puts the water-bath Back stroke and desolvates, take out immediately, put coldly, add 2-10% vanillin glacial acetic acid solution and perchloric acid, shake up, put in the water-bath and heat, take out, use water cooling, add glacial acetic acid, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder respectively and add injection water dissolving or get infusion solutions and concentrate or directly get injection; Put in the separatory funnel, use chloroform extraction 1-5 time, the combined chloroform extracting solution, with distilled water wash 1-5 time, discard chloroform solution, washing liquid and above-mentioned water layer merge, put in the separatory funnel,, merge n-butanol extracting liquid with water saturated n-butanol extraction 1-6 time, add anhydrous sodium sulfate, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, evaporate to dryness, use the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, treat that liquid level is near cotton layer after, use the distilled water eluting, discard washing liquid, use the 40-90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and adds the chloroform dissolving, shakes up, in contrast product solution;
The preparation of need testing solution: get lyophilized powder respectively and add injection water dissolving or get infusion solutions and concentrate or directly get injection; Put in the tool plug test tube, add the 1-3mol/L sulfuric acid solution, shake up, the accurate again chloroform that adds, close plug, jolting, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
10. according to the method for quality control of the ejection preparation of the described treatment tumor of claim 9, it is characterized in that: described method of quality control comprises:
Character:
For injection: product is the clear liquid of light brown;
For infusion solutions: product is yellow clear liquid to light brown;
For lyophilized powder: product is yellow to the loose block of light brown;
Differentiate: (1) is got lyophilized powder 30-100g and is added injection water 5-20ml and make dissolving or get infusion solutions 30-100ml and be concentrated into 5-20ml; Or get injection 5-20ml; Get above-mentioned medicine respectively, add ethanol 20-100ml, stir evenly, filter, precipitate washing with alcohol 1-5 time, precipitate is dissolved with distilled water 2-10ml, get 0.5-3ml and put in the test tube, add 2-10% alpha-Naphthol alcoholic solution 2-10 after the slight fever and drip, shake up, slowly add concentrated sulphuric acid 0.2-2ml along tube wall again, at two liquid junction place displaing amaranth rings;
(2) getting lyophilized powder 200-400g adds injection water 40-60ml and makes dissolving or get infusion solutions 200-400ml and be concentrated into 40-60ml; Or get injection 40-60ml; Get above-mentioned medicine respectively, put in the separatory funnel, with water saturated n-butanol extraction 1-5 time, merge extractive liquid,, put evaporate to dryness in the water-bath, residue dissolves with distilled water 1-10ml, behind the DA-201 resin of internal diameter 1～1.5cm, the long 15cm that adding prepares in advance, filling 12cm, recharge on the DA-201 resin column of 2g neutral alumina water 50-150ml washing, reuse 20-85% methanol solution 30-100ml eluting, collect eluent, evaporate to dryness, residue add methanol 0.5-3ml makes dissolving, as need testing solution; Other gets ginsenoside Re, Rg1, Rb and astragaloside reference substance, adds methanol and makes the mixed solution that every 1ml contains 0.5-3mg, in contrast product solution; According to the thin layer chromatography test, draw each 3-20 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol or acetonitrile: water=1-10: 5-15: 0.5-6: 1-10, lower floor's solution of placing below 10 ℃ is developing solvent, launches, take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ of heating, put under the ultra-violet lamp 200-500nm and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
Check:
PH value: should be 3.0-6.0;
Heavy metal: get lyophilized powder 40-80g and add injection water 10ml and make dissolving or get infusion solutions 2ml or get injection 2ml, put in the crucible, evaporate to dryness in the water-bath is checked according to Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
Pyrogen: get lyophilized powder 100-200g respectively and add injection water 30ml and make dissolving or get infusion solutions 30ml or get injection 30ml; Be diluted to 80-150ml with sodium chloride injection or 5～10% glucose injections,, check according to the Chinese Pharmacopoeia pyrogen test by the dosage of rabbit body weight 1kg injection 10ml, should be up to specification;
Other: injection of the present invention, lyophilized powder or infusion solutions should meet Chinese Pharmacopoeia about the pertinent regulations under the injection item;
Assay: (1) ginsenoside Re
The preparation of reference substance solution: precision takes by weighing ginsenoside Re's reference substance, adds dissolve with methanol and makes and contain ginsenoside Re 1-2mg among every 1ml, shakes up, promptly;
The preparation of standard curve: precision is measured reference substance solution 0,20,40,60,80,100 μ l, put respectively in the 10ml tool plug test tube, putting the water-bath Back stroke desolvates, take out immediately, put cold, add 2-10% vanillin glacial acetic acid solution 0.1-1ml, perchloric acid 0.5-1.5ml, shake up, put in the 40-80 ℃ of water-bath and heated 10-30 minute, take out, with water cooling 0.5-5 minute, add glacial acetic acid 2-10ml, shake up, according to the Chinese Pharmacopoeia spectrophotography, measuring trap at 300-700nm wavelength place, is that vertical coordinate, concentration are abscissa with the trap, the drawing standard curve;
The preparation of need testing solution: get lyophilized powder 60-150g and add injection water 10-30ml and make dissolving or get infusion solutions 50-200ml and be concentrated into 10-30ml; Or get injection 10-30ml; Precision is measured above-mentioned medicine respectively, puts in the separatory funnel, uses chloroform extraction 1-5 time, the combined chloroform extracting solution is used distilled water wash 1-5 time, discards chloroform solution, washing liquid and above-mentioned water layer merge, and put in the separatory funnel, with water saturated n-butanol extraction 1-6 time, merge n-butanol extracting liquid, add anhydrous sodium sulfate 1-10g, stir, put to clarification, n-butyl alcohol liquid is moved in the evaporating dish, wash anhydrous sodium sulfate with n-butyl alcohol, washing liquid is incorporated in the evaporating dish, and evaporate to dryness is used the dissolved in distilled water residue, be added on the macroporous adsorbent resin DA-201 post, after treating that liquid level is near cotton layer,, discard washing liquid with distilled water 100ml eluting, use the 40-90% ethanol elution, collect eluent in evaporating dish, put evaporate to dryness in the water-bath, use the dissolve with methanol residue, move in the 5ml measuring bottle, add methanol to scale, shake up, promptly;
Algoscopy: precision is measured need testing solution 100 μ l, and the method under the preparation of sighting target directrix curve is measured trap from " putting in the 10ml tool plug test tube " in accordance with the law, reads the weight μ g of ginsenoside Re the need testing solution from standard curve, calculates; In this ejection preparation, contain Radix Ginseng in the injection, must not be less than 0.15mg/ml in the ginsenoside Re; Contain Radix Ginseng in the lyophilized powder in the ginsenoside Re, must not be less than 2%; Contain Radix Ginseng in the great transfusion preparation in the ginsenoside Re, must not be less than 0.02mg/ml;
(2) cantharidin is according to the Chinese Pharmacopoeia gas chromatography determination
Chromatographic condition and system suitability test: with carbowax-20M and methyl silicone rubber SE-30 is fixative, and coating concentration is respectively 5-20% and 2-10%, 1-9: 9-1 mixes the dress post; Column temperature is 160 ± 10 ℃; Number of theoretical plate is pressed the cantharidin peak and is calculated, and should be not less than 1500;
The preparation of reference substance solution: get the cantharidin reference substance, the accurate title, decide, and chlorination is copied into the solution that every 1ml contains 0.05-0.1mg, in contrast product solution;
The preparation of need testing solution: get lyophilized powder 30-100g and add injection water 5-20ml and make dissolving or get infusion solutions 30-100ml and be concentrated into 5-20ml; Or get injection 5-20ml; Get above-mentioned medicine respectively, put in the tool plug test tube, add 1-3mol/L sulfuric acid solution 0.5-3ml, shake up, the accurate again 0.5-3ml chloroform that adds, close plug, jolting 1-10 minute, standing demix, centrifugal in case of necessity, get subnatant promptly;
Algoscopy: precision is measured reference substance solution and each 2-15 Ma of need testing solution respectively, and inject gas chromatograph calculates; In this ejection preparation, containing cantharidin in the injection is 0.0005～0.004mg/ml; Containing cantharidin in the lyophilized powder is 0.01～0.1%; Containing cantharidin in the great transfusion preparation is 0.00008～0.0008mg/ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101181339B (en) * | 2007-11-19 | 2011-03-30 | 贵州益佰制药股份有限公司 | Detection method of aidi injection preparations |
| CN102225089A (en) * | 2011-06-07 | 2011-10-26 | 贵州益佰制药股份有限公司 | Pharmaceutical composition for treating cancers, pharmaceutical preparation as well as applications and production methods thereof |
| CN102707008A (en) * | 2012-06-15 | 2012-10-03 | 湖南麓山天然植物制药有限公司 | Detection method of gynostemma total saponin granules |
| CN104950101A (en) * | 2015-07-20 | 2015-09-30 | 石药银湖制药有限公司 | Checking method for Chinese medicine injection trace resin |
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2005
- 2005-12-29 CN CNB2005102008812A patent/CN100487452C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101181339B (en) * | 2007-11-19 | 2011-03-30 | 贵州益佰制药股份有限公司 | Detection method of aidi injection preparations |
| CN102225089A (en) * | 2011-06-07 | 2011-10-26 | 贵州益佰制药股份有限公司 | Pharmaceutical composition for treating cancers, pharmaceutical preparation as well as applications and production methods thereof |
| CN102225089B (en) * | 2011-06-07 | 2012-10-03 | 贵州益佰制药股份有限公司 | Pharmaceutical composition for treating cancers, pharmaceutical preparation as well as applications and production methods thereof |
| CN102707008A (en) * | 2012-06-15 | 2012-10-03 | 湖南麓山天然植物制药有限公司 | Detection method of gynostemma total saponin granules |
| CN104950101A (en) * | 2015-07-20 | 2015-09-30 | 石药银湖制药有限公司 | Checking method for Chinese medicine injection trace resin |
| CN104950101B (en) * | 2015-07-20 | 2016-08-24 | 石药银湖制药有限公司 | A kind of inspection method of Chinese medicine trace resin |
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| CN100487452C (en) | 2009-05-13 |
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