CN1911393A - Injection contg. traditional Chinese medicine, and its quality control method - Google Patents
Injection contg. traditional Chinese medicine, and its quality control method Download PDFInfo
- Publication number
- CN1911393A CN1911393A CN 200510090430 CN200510090430A CN1911393A CN 1911393 A CN1911393 A CN 1911393A CN 200510090430 CN200510090430 CN 200510090430 CN 200510090430 A CN200510090430 A CN 200510090430A CN 1911393 A CN1911393 A CN 1911393A
- Authority
- CN
- China
- Prior art keywords
- solution
- methanol
- ginsenoside
- preparation
- amount
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Steroid Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
A quality control method for the Chinese-medicinal injection prepared from brevis capine, ginseng or red ginseng or pilose asiabell root, ophiopogon root and schisandra fruit includes all or part of the fingerprint test, differential test and content measurement of 4 raw materials.
Description
Technical field
The present invention relates to a kind of method of quality control of the Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis, belong to the field of medical technology.
Background technology
Along with the development of human civilization, the raising of people's living standard, cardiovascular and cerebrovascular diseases such as coronary heart disease, angina pectoris have become human second largest killer.Exploitation heart disease class medicine has become instant challenge of pharmacy industry.The applicant carries out Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis to have developed after the assembly and a kind ofly is prepared into injection by Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis, and it comprises injection, injection powder injection etc.Calculate according to components by weight percent, it by Radix Ophiopogonis 10-500 part, Radix Ginseng 1-300 part, Fructus Schisandrae Chinensis 1-300 part and Herba Erigerontis 50-1000 part through extracting refining and add the injection that suitable adjuvant is made, or the extract that is obtained after extracting by corresponding weight portion medical material is through refining and add the injection that suitable adjuvant is made.For the effective quality of control product, the safety of medicine that satisfy the requirement of producing, guarantees to produce, reliable and curative effect is accurate, the applicant has set up its quality standard.The chemical constituent of known Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis has tens kinds.If its inherent quality is described with one, two kinds of active component, has certain one-sidedness, said nothing of the index components of no efficacy, therefore, the active component of not only having chosen Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis carries out assay, with the quality of how much judging of its content, has more set up finger printing, its material group integral body is controlled, effectively characterized its quality on the whole.Chinese medicine fingerprint is meant chromatograph or spectrographic collection of illustrative plates common, that have distinctive certain class or number constituents in certain Chinese crude drug or the Chinese patent medicine.Do not have under the clear and definite situation in the present stage Effective Components of Chinese Herb overwhelming majority, the quality for effective control Chinese crude drug or Chinese patent medicine has great importance.The Japan main manufacturing enterprise of Chinese prescription medicine just adopts the high-efficiency liquid-phase fingerprint control of quality in enterprises in the eighties in 20th century.Germany, France find that the medical function of Folium Ginkgo extract is extract gained material group's mass action result in the process that Folium Ginkgo extract is developed jointly, and to the quality control of such integral body, also adopt high performance liquid chromatography.In the post medical herbs guide that U.S. FDA is formulated clearly with the method for quality control of finger printing as the compounding substances group.Finger printing has become common recognition at present as Chinese herbal medicine and extraction of substance amount control method thereof.
Summary of the invention
The objective of the invention is by a kind of method of quality control of the Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis is provided, this method of utilizing the inventor to provide, can effectively control the quality of the Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis, concrete guidance is carried out in production to medicine: this method that provides simultaneously is simple, the precision height, favorable reproducibility.
The present invention constitutes like this:
The method of quality control of the Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis comprises following all or part of content:
1) finger printing test, comprise based on the finger printing of Herba Erigerontis composition characteristics based on the finger printing of Radix Codonopsis composition characteristics and based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing and based on the finger printing of Fructus Schisandrae Chinensis composition characteristics;
(2) Herba Erigerontis medical material, Radix Ginseng Rubra or Radix Ginseng or codonopsis pilosula, Radix Ophiopogonis medical material, schisandra chinensis medicinal material, scutellarin, ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the differential test method of all or part of composition in Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B, lobetyolin, atractylenoide etc.;
(3) scutellarin, ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the content test method of all or part of composition such as Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schisandrin, schisantherin B, schisantherin A, deoxyschizandrin, schisandrin B, schisandrin C, total saponins, total lignans, total flavones, total polysaccharides, lobetyolin, atractylenoide.
Described finger printing based on the Herba Erigerontis composition characteristics based on the finger printing of Radix Codonopsis composition characteristics based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing and based on the fingerprint test method of Fructus Schisandrae Chinensis composition characteristics be:
A, employing liquid chromatography test Herba Erigerontis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance scutellarin in an amount of Herba Erigerontis medical material, be diluted to suitable concn with methanol or dissolve with ethanol, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol: 0.005mol/L~2mol/L sodium dihydrogen phosphate (phosphoric acid is regulated pH=2.0~5.0) or 0.2%~5% glacial acetic acid or 0.2%~5% formic acid or 0.2%~3% phosphoric acid solution, gradient elution, flow velocity is that 0.5~2.0ml/min, detection wavelength are one or several in the 190-400nm scope, and column temperature is 20~60 ℃;
(4) based on the formulation of the standard finger-print of Herba Erigerontis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Herba Erigerontis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00;
B, adopt liquid chromatography test Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics be main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Ginseng Rubra or Radix Ginseng and the Radix Ophiopogonis medical material, comprise the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, ophiopogonin D ' in one or more, water or methanol, dissolve with ethanol are diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 20%~99% acetonitrile or 20%~99% methanol: 0.01mol/L~2mol/L sodium dihydrogen phosphate or 0.2%~3% glacial acetic acid or 0.2%~3% formic acid or 0.2%~3% phosphoric acid solution, gradient elution, flow velocity is that 0.5~2.0ml/min, detection wavelength are one or several in the 190-300nm scope, and column temperature is 20~60 ℃;
(4) based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the formulation of standard finger-print: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with said method as Radix Ginseng Rubra or Radix Ginseng in the injection to be measured and Radix Ophiopogonis ingredients fingerprint means of testing;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00;
C, employing liquid chromatography are tested the finger printing based on the Fructus Schisandrae Chinensis composition characteristics:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds water or methanol, dissolve with ethanol or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active product in contrast in an amount of schisandra chinensis medicinal material, comprise in schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schizantherin the second grade one or more, water or methanol, ethanol dilution are as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is acetonitrile or methanol: water or 0.01%~1% phosphoric acid solution or 0.2%~3% glacial acetic acid solution or 0.2%~3% formic acid solution, gradient elution, flow velocity are that 0.5~1.5ml/min, detection wavelength are 203~390nm, and column temperature is 20~50 ℃;
(4) based on the formulation of the standard finger-print of Fructus Schisandrae Chinensis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Fructus Schisandrae Chinensis ingredients fingerprint in the injection to be measured;
(6) with injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.80~1.00;
D, employing liquid chromatography test Radix Codonopsis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that scutellarin, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance in an amount of codonopsis pilosula, comprise in lobetyolin, the atractylenoide one or more, water or methanol, dissolve with ethanol are diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 5%~99%: 95%~5% acetonitrile or methanol-water or 0.01mol/L~2mol/L sodium dihydrogen phosphate or 0.2%~3% glacial acetic acid or 0.2%~3% formic acid or 0.2%~3% phosphoric acid solution, flow velocity is 0.5~2.0ml/min, detect wavelength is that one or several or evaporation photodetector in the 190-300nm scope detects, and column temperature is 20~60 ℃;
(4) based on the formulation of the standard finger-print of Radix Codonopsis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Radix Codonopsis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00.
The method of quality control of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis comprises one or more in the following finger printing:
A, adopt the finger printing of liquid chromatography for measuring based on the Herba Erigerontis composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: it is an amount of that precision takes by weighing scutellarin, adds methanol and make the solution that every 1ml contains 0.1mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is an acetonitrile, Mobile phase B is 0.02moL/L sodium dihydrogen phosphate (25% phosphoric acid is transferred pH=3.5), gradient elution, solvent ratios was from 0 minute to 8 minutes, and the ratio of mobile phase A rises to 30% by 18%, from 8 minutes to 15 minutes, the ratio of acetonitrile rises to 50% by 30%, and from 15 minutes to 18 minutes, the ratio of acetonitrile reduced to 18% by 50%, from 18 minutes to 60 minutes, the ratio of acetonitrile was 18%; Flow velocity is 1.0ml/min, and the detection wavelength is 283 ± 2nm, and column temperature is 40 ℃;
(4) based on the formulation of the standard finger-print of Herba Erigerontis composition characteristics: accurate respectively each the 10 μ l of object of reference solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with the means of testing of said method as Herba Erigerontis ingredients fingerprint in the pulse restoring injection to be measured;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
B, adopt liquid chromatography for measuring based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg
1In right amount, add methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is the 0.05mol/L potassium dihydrogen phosphate, and Mobile phase B is acetonitrile-water (80: 20), gradient elution, solvent ratios was from 0 minute to 5 minutes, the ratio of Mobile phase B is 25%, and from 5 minutes to 40 minutes, the ratio of Mobile phase B rose to 64% by 25%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 64%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 80% by 64%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 25% by 80%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;
(4) based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the formulation of standard finger-print: accurate respectively each the 10 μ l of object of reference solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with said method as Radix Ginseng Rubra or Radix Ginseng in the pulse restoring injection to be measured and Radix Ophiopogonis ingredients fingerprint means of testing;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
C, adopt the finger printing of liquid chromatography for measuring based on the Fructus Schisandrae Chinensis composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the schisandrin reference substance, adds water and make the solution that every 1ml contains 0.1mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is methanol-water, gradient elution, solvent ratios are from 0 minute to 6 minutes, and the ratio of methanol is 68%, from 6 minutes to 8 minutes, the ratio of methanol rises to 76% by 68%, and from 8 minutes to 10 minutes, the ratio of methanol was 76%, from 10 minutes to 15 minutes, the ratio of methanol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methanol was 68%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;
(4) based on the formulation of the standard finger-print of Fructus Schisandrae Chinensis composition characteristics: accurate respectively each the 10 μ l of reference substance solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with the means of testing of said method as Fructus Schisandrae Chinensis ingredients fingerprint in the pulse restoring injection to be measured;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
D, employing liquid chromatography test Radix Codonopsis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that scutellarin, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active atractylenoide in an amount of codonopsis pilosula, be diluted to suitable concn, as object of reference solution with dissolve with methanol;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 67%: 33% methanol-water, and flow velocity is that 1.0ml/min, evaporation photodetector detect, 30 ℃ of drift tube temperatures, and carrier gas flux 2.2L/min, column temperature are 30 ℃;
(4) based on the formulation of the standard finger-print of Radix Codonopsis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Radix Codonopsis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.90~1.00.
(4) based on the formulation of the standard finger-print of Fructus Schisandrae Chinensis composition characteristics: accurate respectively each the 10 μ l of reference substance solution and need testing solution that draw, inject chromatograph of liquid, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with the means of testing of said method as Fructus Schisandrae Chinensis ingredients fingerprint in the pulse restoring injection to be measured.
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00.
The discrimination method of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis is following all or part of content:
The thin layer chromatography discrimination method of Herba Erigerontis medical material, scutellarin in a, the injection:
It is an amount of to get each preparation, adds ethyl acetate or ethanol equal solvent and extracts, and filters, and filtrate volatilizes, and residue is with the dissolving of methanol equal solvent, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Herba Erigerontis control medicinal material, shines medical material solution in pairs with legal system; Get the scutellarin reference substance again, add the methanol equal solvent and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254Lamellae, with ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1~60: 0.2~10: 0.3~5 or benzene or toluene-ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1~30: 0.5~15: 0.05~5: 0.01~3 is developing solvent, launch, take out, dry, spray is with 0.3~10% aluminum chloride ethanol or 0.3~10% aluminum chloride methanol solution, 105 ℃ were dried by the fire 1~15 minute, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of scutellarin in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~95%: 95%~5% methanol or acetonitrile-water or 0.05%~10% glacial acetic acid aqueous solution or 0.02%~2% phosphate aqueous solution or 2%~30%: 2%~30%: 96%~40% methanol or acetonitrile-oxolane-0.02%~2% phosphate aqueous solution or acetonitrile-0.005~0.3moL/L sodium dihydrogen phosphate (1~99% phosphoric acid is transferred pH=2.0~5.0) gradient elution system is a mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
C. the thin layer chromatography discrimination method of schisandra chinensis medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the injection:
It is an amount of to get each preparation, adds chloroform or ether or ethyl acetate or normal hexane or 10~95% ethanol equal solvents and extracts, and filters, and filtrate volatilizes, and residue adds chloroform or the dissolving of ethyl acetate equal solvent, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Fructus Schisandrae Chinensis control medicinal material, shines medical material solution in pairs with legal system; Get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B reference substance again, add chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned ten kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254Lamellae, with petroleum ether (30~60 ℃) or petroleum ether (60~90 ℃)-Ethyl formate or ethyl acetate-formic acid or acetic acid 2~40: 0.2~15: 0.1~5 upper solution or benzene or toluene-ethyl acetate or Ethyl formate 1~30: 0.2~10 or normal hexane-benzene or toluene-Ethyl formate or ethyl acetate-formic acid or acetic acid 0.2~5: 0.5~10: 1~15: 0.1~5 are developing solvent, launch, take out, dry, putting uviol lamp 365nm or 254nm inspects or sprays with 2~20% chromotropic acids-concentrated sulfuric acid solution or 2~20% phosphomolybdic acid ethanol solutions or anisaldehyde sulfuric acid solution, 80 ℃~160 ℃ are dried by the fire the clear or smoked colour developing of iodine to the speckle colour developing, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
The liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in d, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
In e, the injection Radix Ophiopogonis medical material the thin layer chromatography discrimination method:
It is an amount of to get each preparation, adds the n-butyl alcohol equal solvent and extracts, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets control medicinal material Radix Ophiopogonis, adds chloroform-methanol or ethyl acetate or n-butyl alcohol equal solvent and extracts, and filters, and evaporate to dryness, residue add chloroform dissolving, medical material solution in contrast; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned three kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with benzene or toluene or chloroform-methanol or ethanol-glacial acetic acid or formic acid or water 8~300: 0.5~100: 0.01~30 or ethyl acetate or Ethyl formate-pyridine-water 0.2~10: 0.1~5: 0.2~10 is developing solvent, launch, take out, dry, put and inspect under uviol lamp 365nm or the 254nm or spray with 10% sulphuric acid or vanillin reagent or 50% sulphuric acid or 5% vanillin reagent or anisaldehyde reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, the fluorescence speckle that should show same color, negative noiseless;
Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:
It is an amount of to get each preparation, and the water equal solvent extracts back n-butyl alcohol and ether or the extraction of ethyl acetate equal solvent, and the extract evaporate to dryness is with the dissolving of methanol equal solvent, as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ', add the methanol equal solvent respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four liquors, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with ethyl acetate or chloroform-methanol-water 3~50: 1~15: 0.1~5 is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
Ophiopogonin B in g, the injection, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in h, the injection:
It is an amount of to get each preparation, adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, regulates pH to neutral, evaporate to dryness, and residue is with chloroform or the dissolving of ethyl acetate equal solvent, as need testing solution.Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, adds chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four liquid, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with normal hexane or methanol-ethyl acetate or chloroform or Ethyl formate-water 0.2~15: 0.2~40: 0.1~5 is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
The liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, take out, put, transfer pH, extract with the chloroform equal solvent to neutral to room temperature, the extracting solution evaporate to dryness, residue filters with microporous filter membrane with the dissolving of methanol equal solvent, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in j, the injection
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:
It is an amount of to get each preparation, extracts with n-butyl alcohol or ethanol equal solvent, filters, and filtrate is as need testing solution; Get Radix Ginseng Rubra or Radix Ginseng control medicinal material, add the chloroform reflux, extract,, discard chloroform solution, residue adds the water-saturated n-butanol equal solvent and extracts, and extracting solution adds ammonia solution, and divide and get the upper strata, evaporate to dryness, residue dissolves with the methanol equal solvent, in contrast medical material solution; Other gets the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add dissolvings such as methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned 7 kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, lower floor's solution or n-butyl alcohol-ethyl acetate or the Ethyl formate-water 1~10: 0.2~2 placed below 5~40: 10~100: 5~50: 0.2~30 10 ℃ with chloroform-ethyl acetate or Ethyl formate-methanol-water: 1~15 upper strata is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent or 50% sulphate reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color, negative noiseless;
K. ginsenoside Rg in the injection
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds water or methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution for the contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels, 5%~40%: 95%~60% methanol or acetonitrile-water or 0.5%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution or 0.5%~5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5~2.0ml/min, the detection wavelength is in 200~350nm scope or evaporate the photodetector detection, and column temperature is in 20~50 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of lobetyolin in L, the injection:
It is an amount of to get each preparation, is dissolved in water, and with the extraction of n-butyl alcohol equal solvent, extract is concentrated into dried, use dissolve with methanol,, or put in C18 or the C8 solid phase extraction column, use 5%~50% methanol, methanol-eluted fractions successively as need testing solution, collect meoh eluate, be concentrated into 1ml, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Radix Codonopsis control medicinal material, shines medical material solution in pairs with legal system; Other gets lobetyolin's reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, n-butyl alcohol-glacial acetic acid or formic acid or ethanol or dehydrated alcohol-water 1~35: 0.1~10: 0.05~5 are developing solvent, launch, and take out, dry, spray is with 3%~30% ethanol solution of sulfuric acid, and 80~150 ℃ were heated 1~30 minute, and put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the fluorescence speckle of same color, negative noiseless;
The liquid chromatograph discrimination method of lobetyolin in m, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.
The discrimination method of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis is following one or more:
The thin layer chromatography discrimination method of Herba Erigerontis medical material, scutellarin in a, the injection:
It is an amount of to get each preparation, adds ethyl acetate extraction, filters, and filtrate volatilizes, and the residue dissolve with methanol is as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Herba Erigerontis control medicinal material, shines medical material solution in pairs with legal system; Get the scutellarin reference substance again, add the methanol equal solvent and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate, toluene-ethyl acetate-formic acid-water 10: 5: 1: 0.5 is developing solvent, launch, take out, dry, spray is with 3% aluminum chloride alcoholic solution, 105 ℃ were dried by the fire 5 minutes, and put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the same color speckle;
The liquid chromatograph discrimination method of scutellarin in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.1% phosphate aqueous solution is a mobile phase at 20%: 80%, and the detection wavelength is 335nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
C. the thin layer chromatography discrimination method of Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the injection:
It is an amount of to get each preparation, adds chloroform and extracts, and filters, and filtrate volatilizes, and residue adds the chloroform dissolution with solvents, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Fructus Schisandrae Chinensis control medicinal material, shine medical material solution in pairs with legal system, get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B reference substance again, add chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned ten kinds of solution, put in same silica gel G F respectively
254Lamellae, with petroleum ether (30~60 ℃)-upper solution of 15: 5: 1 of Ethyl formate-formic acid is developing solvent, launch, take out, dry, put uviol lamp 254nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless;
The liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in d, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Radix Ophiopogonis in e, the injection:
It is an amount of to get each preparation, adds chloroform-methanol (7: 3) mixed solution and extracts, and filters, and evaporate to dryness, residue add the chloroform dissolving, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets control medicinal material Radix Ophiopogonis, adds chloroform-methanol and extracts, and filters, and evaporate to dryness, residue add chloroform dissolving, medical material solution in contrast; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned three kinds of solution, put in same silica gel G F respectively
254On the lamellae, be at 80: 5: 0.1 developing solvent, launch, inspect under the 254nm with toluene-methanol-glacial acetic acid, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color, negative noiseless;
Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:
It is an amount of to get each preparation, with using n-butanol extraction behind the water dissolution, and the extract evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ', add the methanol equal solvent respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate-methanol-water is developing solvent at 15: 5: 1, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that shows same color, negative noiseless;
Ophiopogonin B in g, the injection, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in h, the injection:
It is an amount of to get each preparation, adds 3% sulphuric acid hydrolysis 4 hours, regulates pH to neutral, evaporate to dryness, and residue dissolves with chloroform, as need testing solution.Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, adds chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four liquid, put respectively on same silica gel g thin-layer plate, with normal hexane-ethyl acetate or-water is developing solvent at 1: 1: 1, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 90 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that shows same color, negative noiseless;
The liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulphuric acid, takes out, and puts to room temperature, transfers pH to neutral, uses chloroform extraction, the extracting solution evaporate to dryness, and the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in j, the injection
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:
Get this product, use n-butanol extraction, filter, filtrate is as need testing solution; Get Radix Ginseng Rubra or Radix Ginseng control medicinal material, add the chloroform reflux, extract,, discard chloroform solution, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; Other gets the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add dissolvings such as methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned 7 kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: lower floor's solution of placing below 1010 ℃ was developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless;
K. ginsenoside Rg in the injection
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of lobetyolin in l, the injection:
It is an amount of to get each preparation, add methanol 25ml, supersound process 30 minutes filters, the filtrate evaporate to dryness, residue adds water 2ml makes dissolving, puts in the C18 solid phase extraction column (500mg is with methanol, each the 10ml prewashing of 20% methanol), use 20% methanol, each 5ml eluting of methanol successively, collect meoh eluate, be concentrated into 1ml, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Radix Codonopsis control medicinal material, shines medical material solution in pairs with legal system; It is an amount of that other gets lobetyolin's reference substance, adds methanol and make every 1ml and contain 1mg solution, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw need testing solution 6 μ l, reference substance solution 2 μ l, put respectively on same high-efficient silica gel G lamellae, with n-butyl alcohol-glacial acetic acid-water (7: 1: 0.5) is developing solvent, launch to take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ were heated 5 minutes, and 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, negative noiseless;
The liquid chromatograph discrimination method of lobetyolin in m, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.
The method of testing of the described Chinese medicine injection agent content made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis should comprise one or more in following:
The assay of scutellarin in a, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~95%: 95%~5% methanol or acetonitrile-water or 0.05%~10% glacial acetic acid aqueous solution or 0.02%~2% phosphate aqueous solution or 2%~30%: 2%~30%: 96%~40% methanol or acetonitrile-oxolane-0.02%~2% phosphate aqueous solution or acetonitrile-0.005~0.3moL/L sodium dihydrogen phosphate (1~99% phosphoric acid is transferred pH=2.0~5.0) gradient elution system is a mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 6.0mg;
Content of total flavone is measured in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol and makes dissolving and fixed to scale in right amount, shakes up, and precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), measure trap at the wavelength place of 335 ± 10nm or get each preparation an amount of, put in the measuring bottle, thin up is to scale, shake up, precision is measured in right amount, puts in the 10ml measuring bottle, adds 50% methanol or water to 5ml, add 5% sodium nitrite solution, 0.3~1ml, shake up, placed 6 minutes, add 10% aluminum nitrate solution, 0.3~1ml, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4~10ml adds 50% methanol or water again to scale, shake up, as need testing solution; With rutin or scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at 500 ± 10nm place, calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the limit of total flavones in rutin or scutellarin, must not be less than 30.0mg;
C. ginsenoside Rg in the injection
1, ginsenoside Rb
1, ginsenoside Re's assay:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution for the contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels, 5%~40%: 95%~60% methanol or acetonitrile-water or 0.5%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution or 0.5%~5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5~2.0ml/min, the detection wavelength is in 200~350nm scope or evaporate the photodetector detection, and column temperature is in 20~50 ℃ of scopes; Calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.0mg, the limit that contains the ginsenoside Re must not be less than 1.5mg, contains ginsenoside Rb
1Limit must not be less than 1.0mg, contain the ginsenoside Rg
1, the ginsenoside Re the limit of summation must not be less than 2.5mg;
D. the assay of total saponins in the injection:
It is an amount of to get each preparation, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.2~4ml, perchloric acid 0.5~5ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, with ice-water bath cooling 2 minutes, precision added glacial acetic acid to 25ml immediately, shake up, as need testing solution; With the ginsenoside Rg
1Or ginsenoside Rb
1Or the product of ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at the wavelength place of 547 ± 10nm, calculates with external standard method or standard curve method, and each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg
1Or ginsenoside Rb
1Or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' meter, must not be less than 18.0mg;
E. total polysaccharides assay in the injection:
It is an amount of that monosaccharide is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg), convert out the content of every bottle of monosaccharide thus;
It is an amount of that total sugar is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 1~4 hour is put coldly, adds 1~2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents in every bottle in the sample with this;
The content of total polysaccharides during the content that deducts monosaccharide with sugar contents promptly gets every bottle;
Each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 70.0mg;
Total lignans assay in f, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use ethyl acetate extraction and extracting solution also, water bath method, and the residue dissolve with methanol is as need testing solution; With schisandrin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), wavelength place at 254 ± 10nm measures trap, calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 4.0mg;
Schisandrin, deoxyschizandrin, schisandrin B assay in g, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains schisandrin must not be less than 0.3mg, and the limit that contains deoxyschizandrin must not be less than 0.3mg; Contain schisandrin B (C
24H
32O
6) limit must not be less than 0.3mg; The limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.9mg;
Ophiopogonin B, ophiopogonin D, ophiopogonin D ' assay in h, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is that 200~410nm or evaporation photodetector detect 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains ophiopogonin B must not be less than 0.05mg, and the limit that contains ophiopogonin D must not be less than 0.05mg; Contain ophiopogonin D ' limit must not be less than 0.01mg; Contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.11mg;
Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin assay in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, take out, put, transfer pH, use chloroform extraction to neutral to room temperature, the extracting solution evaporate to dryness, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is that 200~410nm or evaporation photodetector detect 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains Ruscus aculeatus L. sapogenin must not be less than 0.01mg, and the limit that contains diosgenin must not be less than 0.01mg; The limit that contains ruscogenin must not be less than 0.01mg; The limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.03mg;
The assay of lobetyolin in j, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains lobetyolin must not be less than 0.1mg.
The method of testing of the described Chinese medicine injection agent content made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis is following one or more methods:
The assay of scutellarin in a, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, acetonitrile-0.1% phosphate aqueous solution (20%: 80%) is a mobile phase, the detection wavelength is 335nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 6.0mg;
Content of total flavone is measured in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol and makes dissolving and fixed to scale in right amount, shakes up, and precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), wavelength place at 335nm measures trap, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains the limit of total flavones in scutellarin, must not be less than 30.0mg;
C. ginsenoside Rg in the injection
1, ginsenoside Rb
1, ginsenoside Re's assay:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is in 10~50 ℃ of scopes; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.0mg, the limit that contains the ginsenoside Re must not be less than 0.5mg, contains ginsenoside Rb
1Limit must not be less than 1.0mg, contain the ginsenoside Rg
1, the ginsenoside Re the limit of summation must not be less than 2.5mg;
D. the assay of total saponins in the injection:
It is an amount of to get each preparation, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method, take out immediately, precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml, shake up, heating is 15 minutes in 60 ℃ of water-baths, takes out, immediately with ice-water bath cooling 2 minutes, precision adds glacial acetic acid to 25ml, shaking up, is blank with the retinue solvent, according to spectrophotography, wavelength place at 547nm measures trap, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg
1Meter must not be less than 18.0mg;
E. total polysaccharides assay in the injection:
It is an amount of that monosaccharide is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg), convert out the content of every bottle of monosaccharide thus;
It is an amount of that total sugar is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 1~4 hour is put coldly, adds 1~2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents in every bottle in the sample with this;
The content of total polysaccharides during the content that deducts monosaccharide with sugar contents promptly gets every bottle;
Each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 70.0mg;
Total lignans assay in f, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use ethyl acetate extraction and and extracting solution, water bath method, the residue dissolve with methanol is a blank with the retinue solvent, according to spectrophotography, wavelength place at 254nm measures trap, calculates with one point external standard method, and each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 4.0mg;
Schisandrin, deoxyschizandrin, schisandrin B assay in g, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65: 35, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains schisandrin must not be less than 0.3mg, and the limit that contains deoxyschizandrin must not be less than 0.3mg; Contain schisandrin B (C
24H
32O
6) limit must not be less than 0.3mg; The limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.9mg;
Ophiopogonin B, ophiopogonin D, ophiopogonin D ' assay in h, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water (90: 10) is a mobile phase, detecting wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains ophiopogonin B must not be less than 0.05mg, and the limit that contains ophiopogonin D must not be less than 0.05mg; Contain ophiopogonin D ' limit must not be less than 0.01mg; Contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.11mg;
Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin assay in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulphuric acid, takes out, put to room temperature, transfer pH, use chloroform extraction and extracting solution also to neutral, evaporate to dryness, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains Ruscus aculeatus L. sapogenin must not be less than 0.01mg, and the limit that contains diosgenin must not be less than 0.01mg; The limit that contains ruscogenin must not be less than 0.01mg; The limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.03mg;
The assay of lobetyolin in j, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains lobetyolin must not be less than 0.1mg.
The content test method of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis, assay result according to the breviscapus pulse-engendering injection, can survey composition in its quality standard except that adjuvant, scutellarin, total flavones, ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, total saponins, total lignans, total polysaccharides, schisandrin, deoxyschizandrin, schisandrin B, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the total content of all or part of kind in Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, lobetyolin, atractylenoide etc. accounts for more than 25%;
Compared with prior art, the present invention's quality of the Chinese medicine injection products made with Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis of perfect control more.The Chinese medicine ingredients complexity, if only with wherein one, two kind of composition illustrate its inherent quality, has certain one-sidedness, more can't judge the index components of no drug effect.Therefore the applicant formulated based on the finger printing of Herba Erigerontis composition characteristics and based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing and control the quality of injection based on the finger printing of Fructus Schisandrae Chinensis composition characteristics comprehensively.But because contained complex chemical composition between each medical material in the breviscapus pulse-engendering injection, formulation to finger printing causes interference, cause each several part finger printing feature instability,, just can obtain good finger printing so must control mobile phase isochromatic spectrum condition.That is to say, the finger printing of breviscapus pulse-engendering preparation is not that the finger printing simple superposition of Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and schisandra chinensis medicinal material or preparation is just getable, because four kinds of medical material ingredients interference effect each other in the prescription, cause the part of Herba Erigerontis in the breviscapus pulse-engendering preparation, Radix Codonopsis part, Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis part, Fructus Schisandrae Chinensis part the finger printing characteristic peak change, and have only the condition of the present invention of employing, just can obtain ideal finger printing.
Proof by experiment, method of quality control of the present invention is more effective to the quality control of the Chinese medicine injection products made with Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis, and method precision, stability are all higher.
Experimental example 1 is based on the preparation of the finger printing of Herba Erigerontis composition characteristics
A, experimental apparatus, reagent and sample
Reference substance: scutellarin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
B, chromatographic condition and system suitability experiment
1. the selection of chromatographic column
In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, tried out Zorbax, Inertsil ODS-3 respectively, Diamonsil ODS (is C18,250mm * 4.6mm, 5 μ m) chromatographic column of three kinds of trades mark, result show that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, and wherein Diamonsil ODS chromatographic column separating effect is best, post is imitated the highest, can reach 28000 (calculating with object of reference scutellarin peak).So finally selecting Diamonsil ODS chromatographic column (250 * 4.6mm, 5 μ m) for use is the experimentation post.
2. the selection of mobile phase
Investigated (1) methanol-water (10: 90) in the research process respectively, (2) methanol-0.1% glacial acetic acid (5: 95), (3) (the gradient elution volume proportion is from 0 minute to 8 minutes to methanol-0.5% glacial acetic acid (gradient elution) (4) acetonitrile-0.02moL/L sodium dihydrogen phosphate (25% phosphoric acid is transferred pH=3.5), the ratio of acetonitrile rises to 30% by 18%, from 8 minutes to 15 minutes, the ratio of acetonitrile rises to 50% by 30%, from 15 minutes to 18 minutes, the ratio of acetonitrile reduces to 18% by 50%, from 18 minutes to 60 minutes, the ratio of acetonitrile was 18%) four kinds of flow phase system.The result shows that peak shape is relatively poor under mobile phase (1) condition, separates bad; Under mobile phase (2) condition, peak shape is better, but it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (3) condition, peak shape is better, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.
3. detection wavelength determination
In the research under acetonitrile-0.02moL/L sodium dihydrogen phosphate (25% phosphoric acid transfer pH=3.5) (gradient elution) mobile phase condition, investigated the chromatographic peak situation under different-waveband typical wavelengths 205,230,254,283,325nm respectively, the result shows, chromatographic peak is less 205,230, under the 325nm, 205, baseline drift is serious under the 230nm, 254, baseline is good under the 283nm, and wherein chromatographic peak is more under the 283nm, so finally select for use 283nm as detecting wavelength.
4. instrument, chromatographic column and integral parameter
4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved Agilent1100 series of high efficiency chromatograph of liquid for use, the Chemstation chromatographic work station.Chromatographic column is Diamonsil ODS (250mm * 4.6mm, 5 μ m); 40 ℃ of column temperatures, flow velocity 1.0ml/min.
4.2 integral parameter: Slope Sensitivity:1, peak width:0.05, smallest peaks area are 5% of object of reference (S) peak-to-peak area, minimum peak height be the S peak-to-peak high 5%.So setting can be avoided some very calculating of the chromatographic peak of small size (unimodal area accounts for total peak area less than 0.5%), guarantees the dependency with object of reference simultaneously.
5. the preparation of need testing solution
It is an amount of that precision takes by weighing this product, adds water and make the solution that every 1ml contains 50mg, promptly.
6. the preparation of object of reference solution
Scutellarin is one of Herba Erigerontis main active, and its integral area proportion in finger printing more greatly and more stable is taken into account the research of intermediate and medical material simultaneously, and therefore selected scutellarin is as object of reference.
7. finger printing and technical parameter
Formulate standard finger-print according to 10 batch samples, test sample finger printing and standard finger-print are compared, similarity is all between 0.90~1.00.
Experimental example 2 based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the preparation of finger printing
A, experimental apparatus, reagent and sample
Reference substance: ginsenoside Rg
1: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
B, chromatographic condition and system suitability experiment
1. the selection of chromatographic column
In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, tried out Zorbax, Inertsil ODS-3 respectively, Diamonsil ODS (is C18,4.6mm * 200m, 5 μ m) chromatographic column of three kinds of trades mark, result show that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, and wherein Diamonsil ODS chromatographic column separating effect is best, post is imitated the highest, can reach 5400 (calculating with the object of reference ginsenoside Rg1).So finally selecting Diamonsil ODS chromatographic column (4.6mm * 200mm, 5 μ m) for use is the experimentation post.
2. the selection of mobile phase
Investigated (1) methanol-water (20: 80) in the research process respectively, (2) acetonitrile-water (10: 80),
(3) acetonitrile-water (gradient elution) (4) A is 50mmol.L
-1KH
2PO
4Solution, B are acetonitrile-water (80: 20)
(the gradient elution volume proportion is from 0 minute to 5 minutes, the ratio of Mobile phase B is 25%, from 5 minutes to 40 minutes, the ratio of Mobile phase B rises to 64% by 25%, and from 40 minutes to 50 minutes, the ratio of Mobile phase B was 64%, from 50 minutes to 65 minutes, the ratio of Mobile phase B rises to 80% by 64%, and from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 25% by 80%) four kinds of flow phase system.The result shows that peak shape is relatively poor under mobile phase (1) condition, and it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (2) the mobile phase condition, peak shape is relatively poor, separates bad; (3) under the condition, the peak hangover is serious, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.
3. detection wavelength determination
Be 50mmol.L at A in the research
-1KH
2PO
4Solution, B are under acetonitrile-water (80: 20) (gradient elution) the mobile phase condition, have investigated the chromatographic peak situation under different-waveband typical wavelengths 203,210,230,254 respectively, the result shows, chromatographic peak is more under 203nm, and peak shape is better, so finally select for use 203nm as detecting wavelength.
4. instrument, chromatographic column and integral parameter
4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved Agilent1100 series of high efficiency chromatograph of liquid for use, the Chemstation chromatographic work station.Chromatographic column is Diamonsil ODS (4.6mm * 200mm, 5 μ m); 40 ℃ of column temperatures, flow velocity 1.0ml/min.
4.2 integral parameter: Slope Sensitivity:1, peak width:0.05, smallest peaks area are 5% of object of reference (S) peak-to-peak area, minimum peak height be the S peak-to-peak high 5%.So setting can be avoided some very calculating of the chromatographic peak of small size (unimodal area accounts for total peak area less than 0.5%), guarantees the dependency with object of reference simultaneously.
5. the preparation of need testing solution
It is an amount of that precision takes by weighing this product, adds water and make the solution that every 1ml contains 50mg, promptly.
6. the preparation of object of reference solution
The ginsenoside Rg
1Be one of Radix Ginseng Rubra or Radix Ginseng main active, its integral area proportion in finger printing more greatly and more stable is taken into account the research of intermediate and medical material simultaneously, therefore selected ginsenoside Rg
1As object of reference.
7. finger printing and technical parameter
Formulate standard finger-print according to 10 batch samples, test sample finger printing and standard finger-print are compared, similarity is all between 0.90~1.00.
Experimental example 3 is based on the preparation of the finger printing of Fructus Schisandrae Chinensis composition characteristics
A, experimental apparatus, reagent and sample
Reference substance: schisandrin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
B, chromatographic condition and system suitability experiment
1. the selection of chromatographic column
In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, tried out Zorbax, Inertsil ODS-3 respectively, Diamonsil ODS (is C18,250mm * 4.6mm, 5 μ m) chromatographic column of three kinds of trades mark, result show that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, and wherein Diamonsil ODS chromatographic column separating effect is best, post is imitated the highest, can reach 5400 (calculating with the object of reference schisandrin).So finally selecting Diamonsil ODS chromatographic column (250mm * 4.6mm, 5 μ m) for use is the experimentation post.
2. the selection of mobile phase
(1) (20: 80) have been investigated in the research process respectively, (2) acetonitrile-water (10: 80), (3) (the gradient elution volume proportion is from 0 minute to 6 minutes to acetonitrile-water (gradient elution) (4) methanol-water, the ratio of methanol is 68%, from 6 minutes to 8 minutes, the ratio of methanol rises to 76% by 68%, from 8 minutes to 10 minutes, the ratio of methanol is 76%, from 10 minutes to 15 minutes, the ratio of methanol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methanol was 68%) four kinds of flow phase system.The result shows that peak shape is relatively poor under mobile phase (1) condition, and it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (2) the mobile phase condition, peak shape is relatively poor, separates bad; (3) under the condition, the peak hangover is serious, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.
3. detection wavelength determination
In the research under methanol-water (gradient elution) mobile phase condition, investigated the chromatographic peak situation under different-waveband typical wavelengths 203,210,230,250,300nm respectively, the result shows that chromatographic peak is more under 250nm, peak shape is better, so finally select for use 250nm as detecting wavelength.
4. instrument, chromatographic column and integral parameter
4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved Agilent1100 series of high efficiency chromatograph of liquid for use, the Chemstation chromatographic work station.Chromatographic column is Diamonsil ODS (4.6mm * 200mm, 5 μ m); 30 ℃ of column temperatures, flow velocity 1.0ml/min.
4.2 integral parameter: Slope Sensitivity:1, peak width:0.05, smallest peaks area are 5% of object of reference (S) peak-to-peak area, minimum peak height be the S peak-to-peak high 5%.So setting can be avoided some very calculating of the chromatographic peak of small size (unimodal area accounts for total peak area less than 0.5%), guarantees the dependency with object of reference simultaneously.
5. the preparation of need testing solution
It is an amount of that precision takes by weighing this product, adds water and make the solution that every 1ml contains 50mg, promptly.
6. the preparation of object of reference solution
Schisandrin is one of main active in the Fructus Schisandrae Chinensis, and its integral area proportion in finger printing more greatly and more stable is taken into account the research of intermediate and medical material simultaneously, and therefore selected schisandrin is as object of reference.
7. finger printing and technical parameter
Formulate standard finger-print according to 10 batch samples, test sample finger printing and standard finger-print are compared, similarity is all between 0.90~1.00.
Experimental example 4 is differentiated and assay
The thin layer chromatography discrimination method of Herba Erigerontis in the breviscapus pulse-engendering injection
Feature for outstanding Herba Erigerontis, selected scutellarin as its feature speckle, but owing to there is composition like more, the polar phase close in the medical material with the scutellarin structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition launches scutellarin:
Condition question
Ethyl acetate-formic acid-water (15-5-1) reference substance is expanded to the forward position
Silica gel g thin-layer plate
Ethyl acetate-formic acid-water (15-3-1) reference substance does not separate, and feminine gender has interference
The silica gel H lamellae
Ethyl acetate-acetic acid-water (10-5-1) reference substance is expanded to the forward position
Silica gel g thin-layer plate
Benzene-ethyl acetate-formic acid-water (10-5-1-0.5) reference substance does not separate, and feminine gender has interference
Silica gel g thin-layer plate
Toluene-ethyl acetate-acetic acid-water (10-5-1-0.5) reference substance does not separate, and feminine gender has interference
Silica gel g thin-layer plate
Toluene-Ethyl formate-formic acid-water (13-3-1-0.5) reference substance does not separate, and feminine gender has interference
Silica gel g thin-layer plate
Determine condition: it is clear that toluene-ethyl acetate-acetic acid-water (10-5-1-0.5) separates, and the moderate negative nothing of Rf value is done
Silica gel g thin-layer plate is disturbed
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, with toluene-ethyl acetate-acetic acid-water
(10-5-1-0.5) be developing solvent, with this understanding, the Rf value of scutellarin is moderate, and it is clear to separate with other speckle, and is negative noiseless.
The liquid chromatograph discrimination method of scutellarin in the breviscapus pulse-engendering injection
Feature for outstanding Herba Erigerontis, selected scutellarin as its characteristic component, but owing to there is composition like more, the polar phase close in the medical material with the scutellarin structure, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase scutellarin are separated:
Condition question
Methanol-water (80: 20) appearance time is too fast
Octadecylsilane chemically bonded silica
Acetonitrile-water (40: 60) feminine gender has interference
Octadecylsilane chemically bonded silica
Methanol-0.2% glacial acetic acid aqueous solution (30: 70) eight alkyl silane feminine genders have interference
Bonded silica gel
Methanol-oxolane-0.2% phosphate aqueous solution (14: 14: 72) peak shape is slightly asymmetric
Octadecylsilane chemically bonded silica
There is bifurcated at acetonitrile-oxolane-0.2% phosphate aqueous solution (14: 14: 72) peak,
The dialkyl silane bonded silica gel
Methanol-water (40: 60) feminine gender has interference
Eight alkyl silane bonded silica gels
Acetonitrile-0.1% phosphate aqueous solution (20: 80) retention time is moderate, and the peak is capable sharp-pointed, symmetry,
The octadecylsilane chemically bonded silica feminine gender is noiseless
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-0.1% phosphate aqueous solution (20: 80) is a mobile phase, and with this understanding, the scutellarin retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless.
The thin layer chromatography discrimination method of schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the breviscapus pulse-engendering injection
Feature for outstanding Fructus Schisandrae Chinensis, selected schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B is as its feature speckle, but owing to exist more and schisandrin in the medical material, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B structure is close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B launches:
Condition question
Normal hexane-benzene-Ethyl formate-formic acid (5-5-10-4) silica gel H reference substance is expanded to the forward position
Lamellae
Normal hexane-benzene-Ethyl formate-formic acid (5-5-5-3) silica gel H reference substance does not separate, and feminine gender has interference
Lamellae
So that petroleum ether (30~60 ℃)-(10: reference substance is expanded to the forward position to Ethyl formate-formic acid
10: 3) the upper solution silica gel g thin-layer plate
So that petroleum ether (60~90 ℃)-(15: 5: reference substance did not separate Ethyl formate-formic acid, and feminine gender has interference
1) upper solution silica gel G F
254Lamellae
Toluene-ethyl acetate (10-5) silica gel H lamellae reference substance does not separate, and feminine gender has interference
Toluene-Ethyl formate (15-5) silica gel g thin-layer plate reference substance does not separate, and feminine gender has interference
Determine condition: so that petroleum ether (30~60 ℃)-Ethyl formate-separation is clear, the moderate feminine gender of Rf value is noiseless
The upper solution silica gel G F of formic acid (15: 5: 1)
254Lamellae
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, upper solution with petroleum ether (30~60 ℃)-Ethyl formate-formic acid (15: 5: 1) is developing solvent, with this understanding, the Rf value of schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B is moderate, it is clear to separate with other speckle, negative noiseless.
The liquid chromatograph discrimination method of schisandrin, deoxyschizandrin, schisandrin B in the breviscapus pulse-engendering injection
Feature for outstanding Fructus Schisandrae Chinensis, selected schisandrin, deoxyschizandrin, schisandrin B as its characteristic component, but because have in the medical material that more and schisandrin, deoxyschizandrin, schisandrin B structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase schisandrin, deoxyschizandrin, schisandrin B are separated:
Condition question
Methanol-water (80: 20) appearance time is too fast
Octadecylsilane chemically bonded silica
Acetonitrile-water (40: 60) feminine gender has interference
Octadecylsilane chemically bonded silica
Methanol-0.2% glacial acetic acid aqueous solution (70: 30) eight alkyl silane bonded silica gel feminine genders have interference
Methanol-0.2% phosphate aqueous solution (70: 30) octadecylsilane chemically bonded silica peak shape is slightly asymmetric
Acetonitrile-0.2% phosphate aqueous solution (70: 30) octadecylsilane chemically bonded silica peak shape is slightly asymmetric
Methanol-water (50: 50) feminine gender has interference
Eight alkyl silane bonded silica gels
Methanol-water (65: 35) retention time is moderate, and the peak is capable
Octadecylsilane chemically bonded silica is sharp-pointed, symmetry, and negative do not have
Disturb
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, methanol-water (65: 35) be a mobile phase, and with this understanding, schisandrin, deoxyschizandrin, schisandrin B retention time are moderate, and the peak is capable sharp-pointed, symmetry, feminine gender is noiseless.
The thin layer chromatography discrimination method of Radix Ophiopogonis in the breviscapus pulse-engendering injection
Feature for outstanding Radix Ophiopogonis, selected the Radix Ophiopogonis control medicinal material as its feature speckle, but because have that more structure is close in the medical material, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:
Condition question
Benzene-methanol-glacial acetic acid (50-10-3) silica gel G F
254The lamellae reference substance does not separate, and feminine gender has interference
Toluene-methanol-glacial acetic acid (50-10-3) silica gel H lamellae reference substance does not separate, and feminine gender has interference
Chloroform-ethanol-formic acid (50-20-5) silica gel g thin-layer plate reference substance is expanded to the forward position
Chloroform-methanol-formic acid (50-20-5) silica gel g thin-layer plate reference substance is expanded to the forward position
Ethyl acetate-pyridine-water (5-3-5) silica gel H lamellae reference substance does not separate, and feminine gender has interference
Benzene-ethanol-formic acid (60-10-2) silica gel g thin-layer plate reference substance does not separate, and feminine gender has interference
Determine condition: it is clear that toluene-methanol-glacial acetic acid (80: 5: 0.1) separates, and the moderate feminine gender of Rf value is noiseless
Silica gel G F
254Lamellae
Through screening, determined with silica gel G F
254Lamellae is an immobile phase, is developing solvent with toluene-methanol-glacial acetic acid (80: 5: 0.1), and with this understanding, the Rf value of principal spot is moderate, and it is clear to separate with other speckle, and is negative noiseless.
Ophiopogonin B in the breviscapus pulse-engendering injection, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method
Feature for outstanding ophiopogonin, ophiopogonin B, ophiopogonin D, ophiopogonin D ' have been selected as its feature speckle, but because have in the medical material that more and ophiopogonin B, ophiopogonin D, ophiopogonin D ' structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:
Condition question
Ethyl acetate-methanol-water (10-10-0.5) silica gel g thin-layer plate reference substance is expanded to the forward position
Chloroform-methanol-water (10-10-0.5) silica gel H lamellae reference substance is expanded to the forward position
Ethyl acetate-methanol-water (10-3-0.2) silica gel g thin-layer plate reference substance does not separate, and feminine gender has interference
Chloroform-methanol-water (15-10-2) silica gel G F
254The lamellae reference substance is expanded to the forward position
Ethyl acetate-methanol-water (20-3-1) silica gel G F
254The lamellae reference substance does not separate, and feminine gender has interference
Ethyl acetate-methanol-water (20-8-1) silica gel g thin-layer plate reference substance does not separate, and feminine gender has interference
Determine condition: it is clear to separate with ethyl acetate-methanol-water (15: 5: 1) silicon, and the moderate feminine gender of Rf value is noiseless
Glue G lamellae
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with ethyl acetate-methanol-water (15: 5: 1), with this understanding, the Rf value of ophiopogonin B, ophiopogonin D, ophiopogonin D ' speckle is moderate, and it is clear to separate with other speckle, and is negative noiseless.
Ophiopogonin B in the breviscapus pulse-engendering injection, ophiopogonin D, ophiopogonin D ' the liquid chromatograph discrimination method
Feature for outstanding ophiopogonin, ophiopogonin B, ophiopogonin D, ophiopogonin D ' have been selected as its characteristic component, but because have in the medical material that more and ophiopogonin B, ophiopogonin D, ophiopogonin D ' structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and mobile phase to ophiopogonin B, ophiopogonin D, ophiopogonin D ' separate:
Condition question
Methanol-water (99: 1) octadecylsilane chemically bonded silica appearance time is too fast
Acetonitrile-water (95: 5) appearance time is too fast
Octadecylsilane chemically bonded silica
Methanol-2% glacial acetic acid aqueous solution (95: 5) feminine gender has interference
Eight alkyl silane bonded silica gels
Methanol-0.2% phosphoric acid aqueous acid (95: 5) feminine gender has interference
Octadecylsilane chemically bonded silica
There is bifurcated at acetonitrile-1% glacial acetic acid aqueous solution (95: 5) peak,
Eight alkyl silane bonded silica gels
Acetonitrile-0.5% phosphoric acid aqueous acid (95: 5) feminine gender has interference
Octadecylsilane chemically bonded silica
Acetonitrile-water (90: 10) retention time is moderate, the capable point in peak
Octadecylsilane chemically bonded silica is sharp, and symmetry is negative noiseless
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water (90: 10) be a mobile phase, and with this understanding, ophiopogonin B, ophiopogonin D, ophiopogonin D ' retention time are moderate, and the peak is capable sharp-pointed, symmetry, feminine gender is noiseless.
The thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the breviscapus pulse-engendering injection
Feature for outstanding ophiopogonin unit, selected Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin as its feature speckle, but because have in the medical material that more and Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:
Condition question
Methanol-chloroform-water (5-3-2) silica gel g thin-layer plate reference substance is expanded to the forward position
Normal hexane-ethyl acetate-water (5-3-1) silica gel H lamellae reference substance is expanded to the forward position
Normal hexane-Ethyl formate-water (3-2-2) silica gel G F
254The lamellae reference substance does not separate, and feminine gender has interference
Methanol-ethyl acetate-water (2-1-1) silica gel g thin-layer plate reference substance does not separate, and feminine gender has interference
Methanol-Ethyl formate-water (1-1-1) silica gel H lamellae reference substance does not separate, and feminine gender has interference
Methanol-Ethyl formate-water (2-1.5-0.5) silica gel H lamellae reference substance does not separate, and feminine gender has interference
Determine condition: it is clear that normal hexane-ethyl acetate-water (1: 1: 1) separates, and the moderate feminine gender of Rf value is noiseless
Silica gel g thin-layer plate
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with normal hexane-ethyl acetate-water (1: 1: 1), with this understanding, the Rf value of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is moderate, and it is clear to separate with other speckle, and is negative noiseless.
The liquid chromatograph discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the breviscapus pulse-engendering injection
Feature for outstanding ophiopogonin unit, selected Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin as its characteristic component, but because have in the medical material that more and Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin are separated:
Condition question
Methanol-water (99: 1) appearance time is too fast
Octadecylsilane chemically bonded silica
Acetonitrile-water (95: 5) appearance time is too fast
Octadecylsilane chemically bonded silica
Methanol-2% glacial acetic acid aqueous solution (95: 5) feminine gender has interference
Eight alkyl silane bonded silica gels
Methanol-0.2% phosphoric acid aqueous acid (95: 5) feminine gender has interference
Octadecylsilane chemically bonded silica
There is bifurcated at acetonitrile-1% glacial acetic acid aqueous solution (95: 5) peak,
Eight alkyl silane bonded silica gels
Acetonitrile-0.5% phosphoric acid aqueous acid (95: 5) feminine gender has interference
Octadecylsilane chemically bonded silica
Acetonitrile-water (90: 10) retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless
Octadecylsilane chemically bonded silica
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water (90: 10) is a mobile phase, with this understanding, wheat Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin retention time are moderate, and the peak is capable sharp-pointed, symmetry, negative noiseless.
Ginsenoside Rg in the breviscapus pulse-engendering injection
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method
For the feature of outstanding Radix Ginseng Rubra or Radix Ginseng, selected the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf be as its feature speckle, but owing to exist more and the ginsenoside Rg in the medical material
1, ginsenoside Rb
1, composition like close, the polar phase of ginsenoside Re, ginsenoside Rf's structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:
Condition question
(20: 60: 40: 10) 10 reference substances were expanded to the forward position to chloroform-Ethyl formate-methanol-water
Lower floor's solution silica gel g thin-layer plate of placing below ℃
N-butyl alcohol-Ethyl formate-water (10-1-5) upper solution silica gel H reference substance is expanded to the forward position
Lamellae
(15: 40: 22: 10) 10 reference substances did not separate chloroform-Ethyl formate-methanol-water, and feminine gender has interference
Lower floor's solution silica gel g thin-layer plate of placing below ℃
(10: 40: 15: 5) 10 ℃ of reference substances did not separate chloroform-ethyl acetate-methanol-water, and feminine gender has interference
Following lower floor's solution silica gel g thin-layer plate of Fang Zhiing
N-butyl alcohol-Ethyl formate-water (5-1-5) upper solution reference substance does not separate, and feminine gender has interference
Silica gel g thin-layer plate
N-butyl alcohol-Ethyl formate-water (2-1.5-0.5) upper solution reference substance does not separate, and feminine gender has interference
The silica gel H lamellae
Determine condition: (15: 40: it was clear to separate, and the moderate negative nothing of Rf value is done for chloroform-ethyl acetate-methanol-water
22: 10) lower floor's solution silica gel g thin-layer plate of placing below 10 ℃ disturbs
Through screening, determined with the silica gel g thin-layer plate to be immobile phase, with chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developing solvent, with this understanding, the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf Rf value moderate, it is clear to separate with other speckle, negative noiseless.
Ginsenoside Rg in the breviscapus pulse-engendering injection
1, ginsenoside Rb
1, the ginsenoside Re the liquid chromatograph discrimination method
For the feature of outstanding Radix Ginseng Rubra or Radix Ginseng, selected the ginsenoside Rg
1, ginsenoside Rb
1, the ginsenoside Re is as its characteristic component, but owing to exist more and the ginsenoside Rg in the medical material
1, ginsenoside Rb
1, composition like close, the polar phase of ginsenoside Re's structure, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and mobile phase to the ginsenoside Rg
1, ginsenoside Rb
1, the ginsenoside Re separates:
Condition question
Methanol-water (99: 5) appearance time is too fast
Octadecylsilane chemically bonded silica
Acetonitrile-water (95: 5) appearance time is too fast
Octadecylsilane chemically bonded silica
Methanol-2% glacial acetic acid aqueous solution (80: 20) feminine gender has interference
Eight alkyl silane bonded silica gels
Methanol-0.2% phosphoric acid aqueous acid (80: 20) feminine gender has interference
Octadecylsilane chemically bonded silica
Acetonitrile-1% glacial acetic acid aqueous solution (90: 10) feminine gender has interference
Eight alkyl silane bonded silica gels
Acetonitrile-0.5% phosphoric acid aqueous acid (85: 15) feminine gender has interference
Octadecylsilane chemically bonded silica
Acetonitrile-water is a mobile phase, gradient elution, and solvent ratios is that retention time is moderate, the peak is capable sharp-pointed, symmetry, negative do not have
From 0 minute to 35 minutes, the ratio of acetonitrile was 19%, disturbed
From 35 minutes to 55 minutes, the ratio of acetonitrile was by 19%
Rise to 29%, from 55 minutes to 70 minutes, acetonitrile
Ratio be 29%, from 70 minutes to 100 minutes, second
The ratio of nitrile rises to 40% by 29%
Octadecylsilane chemically bonded silica
Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rises to 40% by 29%, with this understanding, and the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re's retention time is moderate, the peak is capable sharp-pointed, symmetry is negative noiseless.
Experimental example 5 assay project approaches are investigated
One, ginsenoside Rg
1, the ginsenoside Re content
1. instrument and reagent
(1) instrument: Agilent1100 high performance liquid chromatograph, chemstationsys work station.The TU-1800SPC ultraviolet spectrophotometer.
(2) reagent: ginsenoside Rg
1, the ginsenoside Re: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
2. detect the selection of wavelength: precision takes by weighing the ginsenoside Rg
1, the ginsenoside Re, split in the 10ml measuring bottle, add methanol dilution and make the solution that every 1ml contains 0.5mg, scan in the 200-400nm wave-length coverage.The ginsenoside Rg
1, the ginsenoside Re all has absorption maximum at the 203nm place, therefore selects the detection wavelength of 203nm as assay.
3. chromatographic condition: Dikma ODS (4.6mm * 250mm, 5um); Mobile phase: acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 35 minutes, the ratio of acetonitrile is 19%, and from 35 minutes to 55 minutes, the ratio of acetonitrile rose to 29% by 19%, from 55 minutes to 70 minutes, the ratio of acetonitrile is 29%, and from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; The detection wavelength is 203nm; Flow velocity: 1.0ml/min.
4. the preparation of reference substance solution: get the ginsenoside Rg1 and the ginsenoside Re is an amount of, accurately claim surely, add methanol respectively and be diluted to scale, shake up, every 1ml contain ginsenoside Rg1 0.30mg, the solution of ginsenoside Re 0.24mg.
5. the preparation of need testing solution: get the about 0.5g of this product, the accurate title, decide, and puts in the 5ml measuring bottle, adds to flow mutual-assistance dissolving and be diluted to scale, shakes up, promptly.
With this understanding, negative sample ginsenoside Rg in the disturbed specimen not
1, the ginsenoside Re mensuration, and separating degree is good.
6. ginsenoside Rg
1Take by weighing the ginsenoside Rg with ginsenoside Re's linear relationship precision
110.20mg, ginsenoside Re 10.72mg, put altogether in the 10ml measuring bottle, it is fixed to scale to add mobile phase, shakes up, product solution (contains the ginsenoside Rg among every 1ml in contrast
11.02mg, ginsenoside Rb
11.01mg, contain ginsenoside Re 1.072mg), precision is measured 2.5ml, puts in the 10ml measuring bottle, adds mobile phase to scale, shakes up, in contrast the product dilute solution.(contain the ginsenoside Rg among every 1ml
10.255mg, contain ginsenoside Re 0.268mg) and accurate reference substance solution 3 μ l, 6 μ l, the 10 μ l of drawing; Reference substance dilute solution 2 μ l, 5 μ l, 10 μ l; Injecting chromatograph of liquid, is vertical coordinate with the peak area, and ginsenoside Rg1 and ginsenoside Re's amount is figure for abscissa, the drawing standard curve.
Ginsenoside Rg1's linear relationship
Numbering ginsenoside Rg1 (μ g) peak area
1 0.510 150.04
2 1.275 324.27
3 2.550 856.87
4 3.060 1024.83
5 6.120 2098.79
6 10.200 3523.14
Regression equation: Y=351.97X-61.521
The coefficient of determination: r=0.9996
The ginsenoside Rg1 is good in 0.510~10.20 μ g scope internal linear relation.
Ginsenoside Re's linear relationship
Numbering ginsenoside Re (μ g) peak area
1 0.536 129.65
2 1.340 283.75
3 2.680 759.26
4 3.216 902.30
5 6.432 1840.48
6 10.720 3075.59
Regression equation: Y=290.94.97X-43.21
The coefficient of determination: r=0.9995
The ginsenoside Re is good in 0.536~10.72 μ g scope internal linear relation.
7. ginsenoside Rg1 and ginsenoside Re's reference substance dilute solution (contain ginsenoside Rg1 0.255mg under the accurate absorption of the reference substance precision test standard curve item among every 1ml, contain ginsenoside Re 0.268mg) 10 μ l, inject chromatograph of liquid, continuous sample introduction is measured 5 times, investigate the precision of reference substance solution, measurement result sees Table 1.
Table 1 ginsenoside Rg1 and the test of ginsenoside Re's reference substance precision
Test number (TN) | 1 | 2 | 3 | 4 | 5 | Meansigma methods | RSD(%) |
Ginsenoside Rg1's peak area ginsenoside Re peak area | 859.78 746.19 | 847.26 774.10 | 846.53 740.15 | 856.40 736.24 | 845.30 730.54 | 851.054 740.044 | 0.772% 0.135% |
The result shows that ginsenoside Rg1 and ginsenoside Re's reference substance solution precision are good.
8. stability test
8.1. ginsenoside Rg1 and ginsenoside Re's reference substance dilute solution (contain ginsenoside Rg1 0.255mg under the accurate absorption of the reference substance stability test standard curve item among every 1ml, contain ginsenoside Re 0.268mg) 10 μ l, inject chromatograph of liquid, measure at 0,6,12,24,48 hour sample introduction, measurement result sees Table 2.
Table 2 reference substance stability test result
Testing time (h) | 0 | 6 | 12 | 24 | 48 | On average | RSD(%) |
Ginsenoside Rg1's peak area ginsenoside Re peak area | 856.87 759.26 | 861.83 755.05 | 853.35 755.69 | 863.07 759.75 | 860.64 756.70 | 859.152 757.29 | 0.464% 0.279% |
The result shows that ginsenoside Rg1 and ginsenoside Re's reference substance solution have good stability.
8.2. need testing solution stability test precision takes by weighing this product 0.5004g, puts in the 5ml measuring bottle, adds the mutual-assistance dissolving and fixed to scale of flowing, shake up, the accurate 10 μ l that draw inject chromatograph of liquid, measure at 0,6,12,24,48 hour sample introduction respectively, measurement result sees Table 5.
Table 5 need testing solution stability test result
Testing time (h) | 0 | 6 | 12 | 24 | 48 | On average | RSD(%) |
Ginsenoside Rg1's peak area ginsenoside Re peak area | 604.09 284.03 | 618.90 286.06 | 604.90 283.35 | 601.91 282.55 | 599.32 278.76 | 605.824 282.95 | 1.258% 0.947% |
The result shows that need testing solution has good stability.
9. replica test
Get under this product content uniformity item 5 bottles on powder pin, get content, porphyrize is therefrom got about 0.5g, accurately claims surely, puts in the 5ml measuring bottle, adds the mutual-assistance dissolving and shake up to scale surely of flowing, and the accurate 10 μ l that draw inject chromatograph of liquid, measure, promptly.The results are shown in Table 4.
Table 4 replica test
Test number | 1 | 2 | 3 | 4 | 5 | Meansigma methods | RSD(%) |
Ginsenoside Rg1's (mg/ bottle) ginsenoside Re (mg/ bottle) | 1.03 0.59 | 1.02 0.57 | 1.03 0.58 | 1.02 0.58 | 1.02 0.61 | 1.02 0.59 | 0.53 2.59 |
10. average recovery test
Adopt the application of sample absorption method, precision takes by weighing ginsenoside Rg1 10.43mg, and ginsenoside Re 8.72mg puts in the 10ml measuring bottle altogether, adds mobile phase to scale, shakes up, and precision is measured 1ml; Get this product content uniformity (031001 batch) item freeze-dried powder down, get content, mixing is therefrom got about 0.5g, and accurate the title decides, and puts altogether in the 5ml measuring bottle, adds mobile phase to scale, shakes up, and precision is drawn 10 μ l, injects chromatograph of liquid, measures, promptly.The result sees Table 15-50 respectively, table 15-51.
Ginsenoside Rg1's content in the breviscapus pulse-engendering injection: 0.1683%
Ginsenoside Re's content in the breviscapus pulse-engendering injection: 0.0962%
The test of table 5 ginsenoside Rg1 average recovery
Numbering | Test sample weighing (g) | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | Response rate % |
1 2 3 4 5 | 0.6458 0.6659 0.6494 0.6441 0.6435 | 1.087 1.121 1.093 1.084 1.083 | 1.043 1.043 1.043 1.043 1.043 | 2.122 2.151 2.122 2.121 2.111 | 99.23 98.75 98.66 99.42 98.56 |
Ginsenoside Rg1's average recovery rate=98.92%; RSD=0.38%
The test of table 6 ginsenoside Re average recovery
Numbering | Test sample weighing (g) | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | Response rate % |
1 2 3 4 5 | 0.6458 0.6659 0.6494 0.6441 0.6435 | 0.621 0.641 0.625 0.620 0.619 | 0.872 0.872 0.872 0.872 0.872 | 1.474 1.479 1.488 1.488 1.486 | 97.82 96.10 98.97 99.54 99.43 |
Ginsenoside Re's average recovery rate=98.37%; RSD=1.47%
11. ten batches of lab scale sample assays
Ten batches of lab scale samples of table 7 assay
Lot number | 1 | 2 | 3 | 4 | 5 |
Ginsenoside Rg1's (mg/ bottle) ginsenoside Re (mg/ bottle) | 1.03 0.52 | 1.05 0.58 | 1.08 0.56 | 1.03 0.58 | 1.04 0.54 |
Lot number | 6 | 7 | 8 | 9 | 10 |
Ginsenoside Rg1's (mg/ bottle) ginsenoside Re (mg/ bottle) | 1.04 0.57 | 1.06 0.51 | 1.04 0.58 | 1.06 0.55 | 1.04 0.58 |
12. three batches of pilot scale sample sizes are measured
Get three batches in this product sample, press the described method of text and handle, sample introduction is measured, and the results are shown in Table 8.
Table 8 three batch sample assay results
Lot number | Ginsenoside Rg1's (mg/ bottle) | Ginsenoside Re's (mg/ bottle) |
1 2 3 | 1.06 1.09 1.08 | 0.55 0.55 0.52 |
Two, Radix Ginseng total saponins
(1) instrument, reagent
Instrument: the general logical TU-1810SPC ultraviolet/visible spectrophotometer of analysing
SARTORIUS BP211D electronic analytical balance
Reagent: vanillin analytical pure Tianjin recovery fine chemistry industry institute
Methanol chromatographically pure J.T.Baker
Glacial acetic acid analytical pure Shanghai reagent one factory
Chemical plant, prosperous source, perchloric acid analytical pure Tianjin
The pure water WAHAHA
(2) method and result
1. the selection precision that detects wavelength takes by weighing ginsenoside Rg1 6.11mg, put in the 100ml measuring bottle, add an amount of methanol supersound process (power 250W, frequency 33KHz) and make dissolving, add methanol to scale, shake up, precision is measured 1.2ml, puts in the 10ml tool plug test tube water bath method solvent, take out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml shakes up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds, shake up, scan in 700~400nm wave-length coverage, the result shows that the ginsenoside Rg1 has absorption maximum at the 547nm place, blank noiseless, therefore selecting 547nm is the detection wavelength of Radix Ginseng total saponins in the spectrophotometry breviscapus pulse-engendering injection.
2. the investigation precision of linear relationship takes by weighing ginsenoside Rg1's reference substance 6.85mg, put in the 100ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHZ) and make dissolving, take out, put to room temperature, add methanol to scale, shake up, precision measures 0.4,0.8,1.2,1.6,2.0ml, split in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), measure trap at the wavelength place of 547nm.With the trap is vertical coordinate, and ginsenoside Rg1's amount (μ g) is an abscissa, the drawing standard curve.See Table 9
Regression equation Y=0.0049X-0.0077; R=0.999
The ginsenoside Rg1 is linear good in 27.4~137.0 μ g scopes.
Table 9 ginsenoside Rg1 standard curve determination data:
Numbering | Ginsenoside Rg1 (μ g) | Trap |
1 2 3 4 5 | 27.4 54.8 82.2 109.6 137.0 | 0.121 0.261 0.404 0.534 0.656 |
3. precision test precision takes by weighing ginsenoside Rg1's reference substance 6.85mg, puts in the 100ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHZ) makes dissolving, take out, put to room temperature, add methanol to scale, shake up, precision is measured 1.2ml, put in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, press operation under the text algoscopy item, METHOD FOR CONTINUOUS DETERMINATION 5 times.See Table 10
The experiment of table 10 Radix Ginseng total saponins precision
Numbering | 1 | 2 | 3 | 4 | 5 | X is average | RSD% |
Absorbance | 0.429 | 0.426 | 0.426 | 0.425 | 0.424 | 0.426 | 0.44 |
4. stability test
The stability test precision of reference substance solution takes by weighing ginsenoside Rg1's reference substance 7.27g, puts in the 100ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHZ) makes dissolving, take out, put to room temperature, add methanol to scale, shake up, precision is measured 1.2ml, put in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, promptly get and press operation under the text algoscopy item, respectively 0,10,20,40, measure in the time of 60 minutes.See Table 11
Table 11 Radix Ginseng total saponins reference substance solution stability experiment
Time (min) | 0 | 10 | 20 | 40 | 60 | X is average | RSD% |
Absorbance | 0.453 | 0.453 | 0.450 | 0.451 | 0.449 | 0.451 | 0.40 |
The stability experiment of need testing solution is got under this product (031001 batch) content uniformity item 5 bottles on powder pin, get content, porphyrize, precision takes by weighing 114.02mg, puts in the 10ml measuring bottle, adds water and makes dissolving and fixed to scale in right amount, shake up, precision is measured 0.2ml, by operating under the text algoscopy item, measures in the time of 0,10,20,40,60 minute respectively.See Table 12
Table 12 Radix Ginseng total saponins need testing solution stability experiment
Time (min) | 0 | 10 | 20 | 40 | 60 | X is average | RSD% |
Absorbance | 0.345 | 0.344 | 0.340 | 0.341 | 0.341 | 0.342 | 0.63 |
5. replica test is got under this product (031001 batch) content uniformity item 5 bottles on powder pin, gets content, porphyrize, therefrom get about 100mg, put in the 10ml measuring bottle, add water and make dissolving and fixed in right amount to scale, shake up, precision is measured 0.2ml, by operating under the text algoscopy item.See Table 13
Table 13 Radix Ginseng total saponins repeated experiment
Numbering | 1 | 2 | 3 | 4 | 5 | X is average | RSD% |
Content (mg/ bottle) | 22.85 | 22.70 | 22.21 | 21.96 | 22.93 | 22.53 | 1.88 |
6. recovery test adopts the application of sample absorption method, gets under this product (031001 batch) content uniformity item 5 bottles on powder pin, gets content, and porphyrize is therefrom got about 45mg, the accurate title calmly; Other gets the about 2mg of ginsenoside Rg1's reference substance, and accurate the title decides, and puts altogether in the 10ml measuring bottle, add water and make dissolving and fixed in right amount to scale, shake up, precision is measured 0.2ml, puts in the 10ml tool plug test tube, by operating under the text algoscopy item, with the retinue solvent is blank, measures trap in accordance with the law, reads the content of ginsenoside Rg1 the need testing solution from standard curve, calculate, promptly.See Table 14
The content of total saponins in the breviscapus pulse-engendering injection: 3.74%
The experiment of table 14 Radix Ginseng total saponins average recovery
Numbering | Test sample weighing (mg) | Total saponins amount in the test sample (μ g) | Ginsenoside Rg1's addition (μ g) | Measured value (μ g) | The response rate (%) |
1 2 3 4 5 | 40.23 46.30 48.72 45.06 44.91 | 30.09 34.63 36.44 33.70 33.59 | 39.60 37.40 38.20 37.00 37.60 | 69.66 71.20 74.87 71.39 70.24 | 99.92 97.78 100.60 101.86 97.47 |
Average recovery rate=99.53% RSD=1.88%
7. ten batches of lab scale sample assays
Ten batches of lab scale samples of table 15 Radix Ginseng total saponins assay
Lot number | 1 | 2 | 3 | 4 | 5 |
Radix Ginseng total saponins (mg/ bottle) | 22.10 | 22.28 | 22.74 | 22.27 | 22.64 |
Lot number | 6 | 7 | 8 | 9 | 10 |
Radix Ginseng total saponins (mg/ bottle) | 22.47 | 22.69 | 22.43 | 22.48 | 22.57 |
8. three batches of pilot scale sample sizes are measured
Get three batches in this product sample, press the described method of text and handle, sample introduction is measured, and the results are shown in Table 16.
Table 16 three batch sample assay results
Lot number | Radix Ginseng total saponins (mg/ bottle) |
1 2 3 | 23.01 22.62 22.69 |
Three, schisandrin
1. instrument and reagent
(1) instrument: Agilent1100 high performance liquid chromatograph, chemstationsys work station, TU-1800SPC ultraviolet spectrophotometer, AE240 electronic balance.
(2) reagent: schisandrin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute (110857-200203);
2. detect the selection of wavelength
Precision takes by weighing schisandrin reference substance 15.00, puts in the 50ml measuring bottle, adds methanol and is diluted to scale, shakes up, and gets the solution that every 1ml contains schisandrin 0.30mg, in 200-400nm wave-length coverage scanning (scanning spectra is as follows).Schisandrin has absorption maximum at the 250nm place, therefore selects the detection wavelength of 250nm as assay.
3. chromatographic condition: Dikma OHS (4.6 * 250mm, 5um); Mobile phase: methanol-water (65: 35) is a mobile phase; The detection wavelength is 250nm; Flow velocity: 1.0ml/min.
4. system suitability test
4.1 the preparation of reference substance solution
Precision takes by weighing schisandrin reference substance 3.04mg, puts in the 10ml measuring bottle, adds methanol and is diluted to scale, shakes up, and gets the solution that every 1ml contains schisandrin 0.304mg.
4.2 the preparation of need testing solution
Get the about 0.5g of this product, the accurate title, decide, and puts in the 10ml measuring bottle, adds methanol to scale, shakes up, and filters, promptly.
4.3 the preparation of negative need testing solution
Take by weighing the negative sample that does not contain Fructus Schisandrae Chinensis, according to the preparation method preparation of need testing solution, as negative need testing solution.
Accurate respectively reference substance solution, need testing solution, each 10 μ l of negative need testing solution of drawing inject chromatograph of liquid, measure the record chromatogram.As seen from the figure, test sample schisandrin peak separates good, and the schisandrin peak is noiseless in the negative test sample.
5. the linear relationship of schisandrin
Precision takes by weighing schisandrin reference substance 15.06mg, put and add methanol in the 50ml measuring bottle and make dissolving and fixed in right amount to scale, shake up, accurate 1 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l, the 10 μ l of drawing, injecting chromatograph of liquid, is vertical coordinate with the peak area, and the amount of schisandrin (μ g) is figure for abscissa, the drawing standard curve the results are shown in Table 17.
Regression equation: Y=1540.47X+38.26
The coefficient of determination: r=0.998
Schisandrin is good in 0.3012~3.0120 μ g scope internal linear relation.
Table 17 schisandrin linear relationship is investigated
Numbering | Schisandrin (μ g) | Peak area |
1 2 3 4 5 6 | 0.3012 0.6024 1.2048 1.8072 2.4096 3.0120 | 437.66 980.45 1879.71 2957.46 3757.37 4600.71 |
6. precision test
Get schisandrin reference substance solution (0.3012mg/ml) under the standard curve item, the accurate 10 μ l that draw inject chromatograph of liquid, measure 5 times, investigate the precision of reference substance solution, and measurement result sees Table 18.
The test of table 18 schisandrin reference substance precision
Test number (TN) | 1 | 2 | 3 | 4 | 5 | On average | RSD(%) |
Peak area | 4692.34 | 4691.17 | 4698.78 | 4691.29 | 4705.46 | 4695.81 | 0.13 |
The result shows that schisandrin reference substance solution precision is good.
7. stability test
Get schisandrin reference substance solution (0.3012mg/ml) under the standard curve item, the accurate 10 μ l that draw inject chromatograph of liquid, measure at 0,6,12,24,48 hour sample introduction respectively, and measurement result sees Table 19.
Table 19 reference substance solution stability test
Testing time (h) | 0 | 6 | 12 | 24 | 48 | On average | RSD(%) |
Peak area | 4692.34 | 4691.17 | 4698.78 | 4691.29 | 4705.46 | 4695.81 | 0.13 |
The result shows that the schisandrin reference substance solution has good stability.
8. replica test
Get this product (lot number: 031001) 5 bottles on powder pin under the content uniformity item, get content, porphyrize is therefrom got about 0.5g, accurate claim fixed, put in the 10ml measuring bottle, add the about 6ml of methanol, jolting adds methanol to scale, shakes up, filter, the accurate μ l that draws injects chromatograph of liquid, measures, promptly.The results are shown in Table 20.
Table 20 replica test
Numbering | 1 | 2 | 3 | 4 | 5 | On average | RSD(%) |
Schisandrin (mg/ bottle) | 0.272 | 0.260 | 0.271 | 0.262 | 0.0265 | 0.266 | 2.00 |
The result shows that test sample repeatability is good.
9. average recovery test
Adopt the application of sample absorption method, get this product (lot number: 031001) 5 bottles on powder pin under the content uniformity item, get content, porphyrize, therefrom get about 0.25g, the accurate title, decide, and precision takes by weighing schisandrin 2.82mg in addition, puts in the 25ml measuring bottle, add methanol and make dissolving and fixed to scale in right amount, shake up, precision measures 1ml and institute's weighing test sample is put in the 10ml measuring bottle altogether, add the about 6ml of methanol, jolting adds methanol to scale, shake up, filter, the accurate subsequent filtrate 10 μ l that draw, inject chromatograph of liquid, measure, promptly.Measurement result sees Table 21 respectively.
Schisandrin content in the breviscapus pulse-engendering injection: 0.04412%
The test of table 21 average recovery
Numbering | Test sample weighing (g) | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | Response rate % |
1 2 3 4 5 | 0.2808 0.2768 0.2654 0.2593 0.2729 | 0.1239 0.1221 0.1171 0.1144 0.1204 | 0.1128 0.1128 0.1128 0.1128 0.1128 | 0.2353 0.2340 0.2284 0.2260 0.2329 | 98.76 99.20 98.67 98.94 99.73 |
Schisandrin average recovery rate=99.06%; RSD=0.43%
10. ten batches of lab scale sample assays
Get this sample, press the described method of text and handle, sample introduction is measured, and the results are shown in Table 22.
Ten batches of lab scale samples of table 22 schisandrin assay result
Lot number | 1 | 2 | 3 | 4 | 5 |
Schisandrin (mg/ bottle) | 0.258 | 0.264 | 0.261 | 0.263 | 0.267 |
Lot number | 6 | 7 | 8 | 9 | 10 |
Schisandrin (mg/ bottle) | 0.266 | 0.264 | 0.263 | 0.259 | 0.266 |
11. the test agent schisandrin is measured in three batches
Get three batches in this product sample, press the described method of text and handle, sample introduction is measured, and the results are shown in Table 23.
Table 23 three batch sample schisandrin assay results
Lot number | 1 | 2 | 3 |
Schisandrin content (mg/ bottle) | 0.265 | 0.262 | 0.258 |
Four, total flavones
1 instrument and reagent
(1) key instrument:
Ultraviolet spectrophotometer TU-1810SPC Beijing Puxi General Instrument Co., Ltd
Electronic analytical balance BP211D SARTORIUS
Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.
(2) reagent:
Rutin Nat'l Pharmaceutical ﹠ Biological Products Control Institute (lot number: 0080-9705)
Ethanol analytical pure atropic is Fine Chemical Co., Ltd now
Aluminum nitrate analytical pure Tianjin chemical reagent three factories
Sodium nitrite analytical pure Tianjin chemical reagent three factories
Northization glass purchase and sale center, sodium hydroxide analytical pure Tianjin
Control substance of Rutin is got in 2 selections that detect wavelength, operates by the preparation method of text reference substance solution, obtains reference substance solution.Get the breviscapus pulse-engendering injection, operate, obtain need testing solution by the preparation method of need testing solution in the text algoscopy.Draw control substance of Rutin solution, need testing solution, press the text total flavones and measure item method suggested down, in 400~800nm wave-length coverage, scan, the result shows, reference substance solution and need testing solution all have absorption maximum at 510nm, and solvent is noiseless, therefore select the detection wavelength of 510nm as general flavone content in the spectrophotometry breviscapus pulse-engendering injection.
The preparation precision of 3 reference substance solution takes by weighing at the control substance of Rutin 20mg of 120 ℃ of drying under reduced pressure to constant weight, puts in the 100ml measuring bottle, adds an amount of supersound process of 60% alcoholic solution (power 250W, frequency 33KHz) makes dissolving, take out, put, add 60% alcoholic solution to scale to room temperature, shake up, precision is measured 25ml, puts in the 50ml measuring bottle, and thin up is to scale, shake up, promptly get (containing anhydrous rutin 0.1mg among every 1ml).
The about 50mg of this product powder is got in the preparation of 4 need testing solutions, and accurate the title decides, and puts in the 50ml measuring bottle, adds an amount of supersound process of 60% alcoholic solution (power 250W, frequency 33KHz) and makes dissolving, takes out, and puts to room temperature, adds 60% alcoholic solution to scale, shakes up, promptly.
5 algoscopy precisions are measured reference substance solution 3.0ml, need testing solution 1.0ml, split in the 10ml measuring bottle, respectively add 30% alcoholic solution and make into 5.0ml, precision adds sodium nitrite solution (1 → 20) 0.3ml and shakes up, and places 6 minutes, add aluminum nitrate solution (1 → 10) 0.3ml again, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4.0ml, add water to scale again, shaking up, placed 15 minutes, is blank with the retinue solvent, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 B), wavelength place at 510nm measures trap respectively, calculates, promptly.
The preparation precision of 6 standard curves is measured control substance of Rutin solution (C=0.1064mg/ml) 0.0ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml 5.0ml splits in the 10ml measuring bottle, add 30% alcoholic solution respectively and make into 5.0ml, precision adds sodium nitrite solution (1 → 20) 0.3ml, shakes up, and places 6 minutes, add aluminum nitrate solution (1 → 10) 0.3ml again, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4.0ml, add water to scale again, shaking up, placed 15 minutes, is blank with the retinue solvent, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 B), measuring trap at 510nm wavelength place, is vertical coordinate with the trap, and concentration (mg/ml) is figure for abscissa, the drawing standard curve sees Table 24.
Table 24 total flavones standard curve determination result
Numbering | Rutin concentration (mg/ml) | Trap |
1 2 3 4 5 6 | 0.000 0.0110 0.0210 0.0320 0.0430 0.0530 | 0.000 0.119 0.226 0.343 0.468 0.572 |
Regression equation: Y=10.818x-0.0005;
Correlation coefficient: γ=0.9999;
The result shows: rutin is good in 0.0110~0.0530mg/ml scope internal linear.
As calculated, the standard curve of rutin is crossed initial point, therefore selects for use one point external standard method to measure content of total flavone.
The accurate absorption of 7 precision test control substance of Rutin solution (concentration: 0.1064mg/ml) 3.0ml, put in the 10ml measuring bottle, press the method under the text algoscopy item, from " add 30% alcoholic solution and make into 5.0ml ", operation is in accordance with the law measured trap at the wavelength place of 510nm, measures 5 times.The results are shown in Table 25.
The test of table 25 precision
Numbering | 1 | 2 | 3 | 4 | 5 | RSD(%) |
Trap | 0.343 | 0.343 | 0.342 | 0.340 | 0.337 | 0.75 |
The result shows that reference substance solution precision is good.
8 stability tests
8.1 the accurate control substance of Rutin solution (concentration: 0.1064mg/ml) 3.0ml of drawing of reference substance solution stability test, put in the 10ml measuring bottle, press the method under the text algoscopy item, from " add 30% alcoholic solution and make into 5.0ml ", operation in accordance with the law, wavelength place at 510nm measures trap, respectively at 0,5,10,15,30 minute replication once.The results are shown in Table 26.
Table 26 reference substance solution stability test
Time (min) | 0 | 5 | 10 | 15 | 30 | RSD(%) |
Trap | 0.343 | 0.343 | 0.342 | 0.340 | 0.337 | 0.75 |
The result shows that reference substance solution has good stability.
8.2 the accurate need testing solution 1.0ml that draws of need testing solution stability test, put in the 10ml measuring bottle, press the method under the text algoscopy item, from " add 30% alcoholic solution and make into 5.0ml ", operation in accordance with the law, measure trap at the wavelength place of 510nm, respectively 0,5,10,15,30min measures trap, the results are shown in Table 27.
Table 27 need testing solution stability test
Time (min) | 0 | 5 | 10 | 15 | 30 | RSD(%) |
Trap | 0.377 | 0.376 | 0.374 | 0.372 | 0.369 | 0.86 |
The result shows that need testing solution is good at the 30min internal stability.
9 replica tests are got the about 50mg of this product powder (totally 5 parts), accurate claim surely, by operating under the preparation of text need testing solution and the algoscopy item, the results are shown in Table 28.
Table 28 replica test
Time (min) | 0 | 5 | 10 | 15 | 30 | Average | RSD(%) |
Content (mg/ bottle) | 15.14 | 15.36 | 15.42 | 15.51 | 15.28 | 15.33 | 0.23 |
The result shows that repeatability is good.
The application of sample absorption method is adopted in the test of 10 average recoveries; get the about 25mg of this product powder (totally 6 parts), and accurate the title decides, and splits in the 50ml measuring bottle; precision takes by weighing through the control substance of Rutin 10.37mg of 120 ℃ of drying under reduced pressure to constant weight; put in the 10ml measuring bottle, adds an amount of supersound process of 60% ethanol and makes dissolving, takes out; put to room temperature; shake up to scale with 60% ethanol is fixed, therefrom precision is measured 0.7ml respectively; 0.85ml; 1.0ml (each 2 parts); split in the above-mentioned 50ml measuring bottle; add an amount of supersound process of 60% alcoholic solution (power 250W, frequency 33KHz) and make dissolving, take out; put to room temperature; be diluted to scale with 60% alcoholic solution, shake up, and filtration; precision is measured subsequent filtrate 1.0ml; split in the 10ml measuring bottle is according to the method under the text algoscopy item, from " adding 30% alcoholic solution and make into 5.0ml " rise, operation in accordance with the law, wavelength place at 510nm measures trap, calculates, promptly.The results are shown in Table 29.
Content of total flavone is in the breviscapus pulse-engendering injection: 3.458%.
The test of table 29 average recovery
Numbering | Test sample sample weighting amount (mg) | General flavone content in the test sample (mg) | Rutin addition (mg) | Measured value (mg) | The response rate (%) |
1 2 3 4 5 6 | 25.29 25.33 24.97 25.64 25.32 24.86 | 0.875 0.876 0.863 0.887 0.876 0.860 | 0.726 0.726 0.881 0.881 1.037 1.037 | 1.568 1.577 1.717 1.764 1.922 1.894 | 95.45 96.56 96.94 99.55 100.87 99.71 |
Average recovery rate=98.17%, RSD=0.22%.
The assay of 11 samples is got this product three batch samples, and according to the preparation of text need testing solution and the operation under the algoscopy item, working sample content the results are shown in Table 30.
Table 30 content of total flavone is measured
Lot number | General flavone content (mg/ bottle) | Measure number of times (n) | RSD(%) |
1 2 3 | 16.49 18.51 17.94 | 5 3 3 | 0.23 1.00 0.67 |
Five, scutellarin
1 instrument and reagent
1.1 key instrument:
High performance liquid chromatograph LC-2010AHT SHIMADZU
Ultraviolet/general all purpose instrument the company limited of analysing in visible spectrophotometer TU-1810SPC Beijing
Electronic analytical balance BP211D SARTORIUS
Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.
1.2 reagent:
Scutellarin Nat'l Pharmaceutical ﹠ Biological Products Control Institute
Methanol analytical pure Beijing Chemical Plant
Acetonitrile chromatographically pure Di Ma company
The pure Beijing of phosphate analysis chemical reagents corporation
2 scutellarins provide scutellarin by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and measuring purity through high performance liquid chromatography (normalization method) is 96.40%, meet assay with reference substance requirement (in mensuration, converting according to 96.40% purity).
It is an amount of that the scutellarin reference substance is got in 3 selections that detect wavelength, and accurate the title decides, and adds methanol and makes the solution that every 1ml contains 0.0463mg, in the interscan of 190~400nm wave-length coverage.The result shows that scutellarin has absorption maximum at 284nm and 335nm place, according to " the Sanitation Ministry medicine standard Chinese traditional patent formulation preparation " relevant kind and in conjunction with bibliographical information, selects the detection wavelength of 335nm as the scutellarin assay.。
4 chromatographic conditions
Chromatograph: SHIMADZU LC-2010AHT;
Chromatographic column: Diamonsil ODS 250mm * 4.6mm 5 μ m;
Mobile phase: acetonitrile-0.1% phosphoric acid (20: 80);
Flow velocity: 1.0ml/min;
Column temperature: 30 ℃;
Sample size: 10 μ l;
Detect wavelength: 335nm.
It is an amount of that the preparation precision of reference substance solution takes by weighing the scutellarin reference substance, adds methanol and make the solution that every 1ml contains 0.05mg, promptly.
The about 40mg of this product powder is got in the preparation of need testing solution, the accurate title, decide, put in the 25ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min, take out, put to room temperature, it is fixed to scale to add methanol, shakes up, filter with microporous filter membrane (0.45 μ m), get subsequent filtrate as need testing solution.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
Obtain scutellarin reference substance, test sample chromatogram according to above-mentioned chromatographic condition, its number of theoretical plate n is all greater than 5000, and the scutellarin chromatographic peak separates clear complete with close peak, and separating degree is all greater than 1.5, and solvent is noiseless.
The about 40mg of this product powder (totally 6 parts) is got in the selection of 5 extraction times, and accurate the title decides, and splits in the 25ml measuring bottle, it is an amount of to add methanol, respectively supersound process (power 250W, frequency 33KHz) 5,10,20min, take out, put, add methanol to scale to room temperature, shake up, filter with microporous filter membrane (0.45 μ m), the accurate subsequent filtrate 10 μ l that draw inject chromatograph of liquid, measure, promptly.See Table 31.
Table 31 extraction time investigation
Extraction time (min) | Content (%) |
5 10 20 | 3.54 3.51 3.51 |
The result shows that supersound process 5min can extract fully.
6 linear relationships investigation precision is measured scutellarin reference substance solution (C=0.978mg/ml) 0.0,0.2,0.4,0.6,0.8,1.0ml, split in the 10ml measuring bottle, be diluted to scale with methanol, shake up, the therefrom accurate respectively 10 μ l that draw, inject chromatograph of liquid, measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D).With the peak area is vertical coordinate, and the amount of scutellarin (μ g) is the abscissa mapping, and the drawing standard curve sees Table 32.
Table 32 scutellarin standard curve determination result
Numbering | Scutellarin amount (μ g) | Conversion back (μ g) | Peak area |
1 2 3 4 5 6 | 0.0000 0.1956 0.3912 0.5868 0.7824 0.9780 | 0.0000 0.1886 0.3771 0.5657 0.7542 0.9428 | 0 603722 1224078 1835374 2505678 3038182 |
Regression equation: Y=3259091.50x-1830.06
Correlation coefficient: γ=0.9999
The result shows: scutellarin is good in 0.1886 μ g~0.9428 μ g scope internal linear.
As calculated, the standard curve of scutellarin is crossed initial point, therefore selects for use one point external standard method to measure the content of scutellarin.
Accurate scutellarin reference substance solution (0.0489mg/ml) the 10 μ l that draw of 7 precision experiment inject chromatograph of liquid, measure 5 times, investigate reference substance solution precision, and measurement result sees Table 33.
The test of table 33 precision
Test number (TN) | 1 | 2 | 3 | 4 | 5 | Meansigma methods | RSD(%) |
Peak area | 1572015 | 1570202 | 1566206 | 1559384 | 1557017 | 1564965 | 0.42 |
The result shows that reference substance solution precision is good.
8 stability experiments
8.1 accurate scutellarin reference substance solution (0.0489mg/ml) the 10 μ l that draw of the stability experiment of reference substance solution inject chromatograph of liquid, inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively, measurement result sees Table 15.
Table 34 reference substance solution stability test
Time (h) | 0 | 2 | 4 | 8 | 24 | Meansigma methods | RSD(%) |
Peak area | 1572015 | 1570202 | 1566206 | 1559384 | 1557017 | 1564965 | 0.42 |
The result shows that 24 hours internal stabilities of reference substance solution are good.
8.2 accurate need testing solution (1.4984mg/ml) the 10 μ l that draw of the stability experiment of need testing solution inject chromatograph of liquid, measure at 0,2,4,8,24 hour sample introduction respectively, the results are shown in Table 35.
Table 35 need testing solution stability experiment
Time (h) | 0 | 2 | 4 | 8 | 24 | Meansigma methods | RSD(%) |
Peak area | 1801360 | 1784028 | 1791959 | 1813617 | 1798693 | 1797931 | 0.61 |
The result shows that 24 hours internal stabilities of need testing solution are good.
9 repeated experiments are got this product, by operating under the preparation of text need testing solution and the algoscopy item, the results are shown in Table 36.
Table 36 repeated experiment
Time (h) | 0 | 2 | 4 | 8 | 24 | Meansigma methods | RSD(%) |
Content (mg/ bottle) | 3.46 | 3.50 | 3.51 | 3.49 | 3.49 | 3.49 | 0.56 |
The application of sample absorption method is adopted in the experiment of 10 average recoveries; get the about 20mg of this product powder (030201 batch) (totally 6 parts); the accurate title decides; split in the 25ml measuring bottle; accurate respectively scutellarin reference substance solution (C=0.978mg/ml) 0.5ml, 0.6ml and the 0.7ml (each two parts) of adding; add an amount of supersound process of methanol (power 250W, frequency 33KHz) 5min, take out; put to room temperature; add methanol to scale, shake up, use microporous filter membrane (0.45 μ m) to filter; the accurate subsequent filtrate 10 μ l that draw; inject chromatograph of liquid, mensuration, promptly.The results are shown in Table 37.
The content of scutellarin is in the breviscapus pulse-engendering injection: 3.49%.
The experiment of table 37 average recovery
Numbering | Test sample sample weighting amount (mg) | Pure product amount (mg) in the test sample | Scutellarin addition (mg) | Conversion back (mg) | Measured value (mg) | The response rate (%) |
1 2 3 4 5 6 | 16.55 16.53 16.87 16.22 16.26 16.13 | 0.5776 0.5769 0.5888 0.5661 0.5675 0.5629 | 0.4890 0.4890 0.5868 0.5868 0.6846 0.6846 | 0.4714 0.4714 0.5657 0.5657 0.6600 0.6600 | 1.0478 1.0444 1.1516 1.1279 1.2248 1.2178 | 99.75 99.17 99.49 99.31 99.59 99.23 |
Average recovery rate=99.42%, RSD=0.23%
11 sample sizes are measured and are got this product three batch samples, according to the preparation of text need testing solution and the operation under the algoscopy item, the results are shown in Table 38.
Table 38 scutellarin assay
Lot number | Scutellarin content (mg/ bottle) | Measure number of times (n) | RSD(%) |
1 2 3 | 3.49 3.49 3.51 | 5 3 3 | 0.56 0.77 0.45 |
Six, total lignans
1 instrument and reagent
(1) key instrument:
Ultraviolet spectrophotometer TU-1810SPC Beijing Puxi General Instrument Co., Ltd
Electronic analytical balance BP211D SARTORIUS
Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.
(2) reagent:
Schisandrin Nat'l Pharmaceutical ﹠ Biological Products Control Institute
Ethanol analytical pure atropic is Fine Chemical Co., Ltd now
The schisandrin reference substance is got in 2 selections that detect wavelength, operates by the preparation method of text reference substance solution, obtains reference substance solution.Get the breviscapus pulse-engendering injection, operate, obtain need testing solution by the preparation method of need testing solution in the text algoscopy.Draw the schisandrin reference substance solution, need testing solution, press the text total lignans and measure item method suggested down, in 200~400nm wave-length coverage, scan, the result shows, reference substance solution and need testing solution all have absorption maximum at 250nm, and solvent is noiseless, therefore select the detection wavelength of 250nm as total lignans content in the spectrophotometry breviscapus pulse-engendering injection.
The preparation precision of 3 reference substance solution takes by weighing at the schisandrin reference substance 15mg of 60 ℃ of drying under reduced pressure to constant weight, put in the 50ml measuring bottle, add an amount of supersound process of methanol solution (power 250W, frequency 33KHz) makes dissolving, take out, put, add methanol solution to scale to room temperature, shake up, promptly get (containing schisandrin 0.3mg among every 1ml).
The preparation precision of 4 standard curves is measured reference substance solution (C=0.2908mg/ml) 0.1ml, 0.2ml, 0.4ml, 0.6ml 0.8ml, 1.0ml split in the 10ml measuring bottle, adding methanol to scale, shake up, is blank with the retinue solvent, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 B), measuring trap at 250nm wavelength place, is vertical coordinate with the trap, and concentration (μ g/ml) is figure for abscissa, the drawing standard curve sees Table 39.
Table 39 total lignans standard curve determination result
Numbering | Schisandrin concentration (μ g/ml) | Trap |
1 2 3 4 5 6 | 2.908 5.816 11.632 17.448 23.264 29.080 | 0.118 0.245 0.491 0.732 0.971 1.217 |
Regression equation: Y=0.0418x+0.0003:
Correlation coefficient: γ=0.9999;
The result shows: schisandrin is good in 2.908~29.080g/ml scope internal linear.
As calculated, the standard curve of schisandrin is crossed initial point, therefore selects for use one point external standard method to measure the content of total lignans.
The accurate absorption of 7 precision test schisandrin reference substance solution (concentration: 0.2908mg/ml) 0.6ml, put in the 10ml measuring bottle, press the method under the text algoscopy item, measure 5 times.The results are shown in Table 40.
The test of table 40 precision
Numbering | 1 | 2 | 3 | 4 | 5 | RSD(%) |
Trap | 0.732 | 0.733 | 0.731 | 0.734 | 0.732 | 0.56 |
The result shows that reference substance solution precision is good.
8 stability tests
8.1 the accurate absorption of reference substance solution stability test schisandrin reference substance solution (concentration: 0.2908mg/ml) 0.6ml, put in the 10ml measuring bottle, press the method under the text algoscopy item, respectively once at 0,5,10,15,30 minute replication.The results are shown in Table 41.
Table 41 reference substance solution stability test
Time (min) | 0 | 5 | 10 | 15 | 30 | RSD(%) |
Trap | 0.732 | 0.733 | 0.731 | 0.734 | 0.732 | 0.56 |
The result shows that reference substance solution has good stability.
8.2 the accurate need testing solution 1.0ml that draws of need testing solution stability test puts in the 10ml measuring bottle, it is fixed to scale to add methanol, measures trap at the wavelength place of 250nm, respectively 0,5,10,15,30min measures trap, the results are shown in Table 42.
Table 42 need testing solution stability test
Time (min) | 0 | 5 | 10 | 15 | 30 | RSD(%) |
Content (mg/ bottle) | 2.12 | 2.16 | 2.14 | 2.18 | 2.15 | 0.63 |
The result shows that need testing solution is good at the 30min internal stability.
9 replica tests are got this product powder approximately, by operating under the preparation of text need testing solution and the algoscopy item, the results are shown in Table 43.
Table 43 replica test
Time (min) | 0 | 5 | 10 | 15 | 30 | Average | RSD(%) |
Content (mg/ bottle) | 2.17 | 2.15 | 2.14 | 2.16 | 2.17 | 2.16 | 1.13 |
The result shows that repeatability is good.
The application of sample absorption method is adopted in the test of 10 average recoveries, gets the about 75mg of this product powder (totally 6 parts), and accurate the title decides, split in the 50ml measuring bottle, precision is measured reference substance solution (C=0.2908mg/ml) 0.8ml, 1.1ml, 1.2ml, split in the above-mentioned 50ml measuring bottle, add an amount of supersound process of methanol solution (power 250W, frequency 33KHz) and make dissolving, take out, put, be diluted to scale with methanol solution to room temperature, shake up, filter, measure trap at the wavelength place of 250nm, calculate, promptly.The results are shown in Table 44.
The content of total lignans is in the breviscapus pulse-engendering injection: 0.421%.
The test of table 44 average recovery
Numbering | Test sample sample weighting amount (mg) | Total lignans content (mg) in the test sample | Schisandrin addition (mg) | Measured value (mg) | The response rate (%) |
1 2 3 4 5 6 | 75.64 78.59 80.12 76.28 73.96 74.25 | 0.3184 0.3309 0.3373 0.3211 0.3114 0.3126 | 0.2326 0.2326 0.3199 0.3199 0.349 0.349 | 0.5429 0.5556 0.6437 0.6409 0.6481 0.6603 | 96.50 96.62 95.78 99.96 96.48 99.63 |
Average recovery rate=97.49%, RSD=1.13%.
The assay of 11 samples is got this product three batch samples, and according to the preparation of text need testing solution and the operation under the algoscopy item, working sample content the results are shown in Table 45.
The assay of table 45 total lignans
Lot number | Total lignans content (mg/ bottle) | Measure number of times (n) | RSD(%) |
1 2 3 | 2.64 2.38 2.59 | 3 3 3 | 0.58 1.13 2.07 |
Seven, total polysaccharides
Monosaccharide is got under the content uniformity item 5 bottles of this product, gets content, mixing, therefrom get about 500mg, the accurate title, decide, and puts in the iodine flask, and adding distil water 60ml makes dissolving, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose (C of 9.008mg
6H
12O
6), convert out the content of every bottle of monosaccharide thus.
Total sugar is got under the content uniformity item 5 bottles of this product, gets content, mixing, therefrom get about 500mg, the accurate title, decide, and puts in the iodine flask, adding distil water 20ml makes dissolving, adds dilute sulfuric acid 25ml, reflux 4 hours, put coldly, add 1~2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose (C of 9.008mg
6H
12O
6), calculate sugar contents in every bottle in the sample with this.
The standard curve precision takes by weighing 105 ℃ of anhydrous glucose reference substance 50.24mg that are dried to constant weight, 101.87mg, 149.58mg, 201.36mg, 252.81mg, 300.79mg, split in the iodine flask, adding distil water 60ml makes dissolving, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose (C of 9.008mg
6H
12O
6).Consuming volumetric solution volume (ml) with sample is abscissa, is that vertical coordinate is figure with the amount (mg) of anhydrous glucose, the drawing standard curve.See Table 46.
Table 46 anhydrous glucose standard curve
Numbering | The sample volumetric solution consumes volume (ml) | Anhydrous glucose (mg) |
1 2 3 4 5 6 | 5.65 11.25 16.85 22.65 28.25 33.74 | 50.24 101.87 149.58 201.36 252.81 300.79 |
Repeated experiment is got this product under the content uniformity item, gets content, and mixing is therefrom got about 500mg (5 parts), presses operation under the text algoscopy item, measures, promptly.Measurement result sees Table 47.
Table 47 repeated experiment
Numbering | Weighing (mg) | Total sugar (mg/ bottle) | Monosaccharide (mg/ bottle) | Total polysaccharides (mg/ bottle) |
12345 average RSD (%) | 510.74 500.39 503.61 500.78 504.92 - - | 189.26 187.63 185.22 186.64 190.15 187.78 1.05 | 110.74 109.31 105.25 106.37 109.59 108.25 2.15 | 78.52 78.32 79.97 80.27 80.56 79.53 1.30 |
The application of sample absorption method is adopted in response rate experiment, gets this product under the content uniformity item (040201 batch), gets content, and mixing is therefrom got about 250mg (6 parts), splits in the iodine flask; Be taken at 105 ℃ of about 45mg of anhydrous glucose that are dried to constant weight, the accurate title, decide, split in the above-mentioned iodine flask, adding distil water 60ml makes dissolving, and hydro-oxidation sodium test solution is to neutral, and precision adds iodine liquid (0.1mol/L) 25ml, shake up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank assay, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose (C of 9.008mg
6H
12O
6).The results are shown in Table 48.
Breviscapus pulse-engendering injection contents of monosaccharides: 108.25mg/ bottle.
The test of table 48 anhydrous glucose average recovery
Numbering | Test sample weighing (mg) | Monosaccharide amount (mg) in the test sample | Anhydrous glucose addition (mg) | Measured value (mg) | The response rate (%) |
1 2 3 4 5 6 | 252.46 247.39 250.61 255.07 252.49 250.18 | 44.61 43.72 44.29 45.08 44.62 44.21 | 40.39 44.28 45.71 42.66 43.92 44.57 | 83.67 87.41 88.96 86.36 87.18 88.25 | 96.71 98.67 97.72 96.71 96.90 98.81 |
Average recovery rate=97.59%; RSD=0.99%.
The assay of sample is got in this product three batches of test agents, presses the preparation and the operation down of algoscopy item of text need testing solution, the results are shown in Table 49.
Table 49 total polysaccharides assay result
Lot number | Total polysaccharides content (mg/ bottle) |
1 2 3 | 74.12 78.36 70.58 |
The specific embodiment
Embodiment 1 finger printing
A, employing liquid chromatography test Herba Erigerontis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance scutellarin in an amount of Herba Erigerontis medical material, be diluted to suitable concn with methanol or dissolve with ethanol, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol: 0.005mol/L~2mol/L sodium dihydrogen phosphate (phosphoric acid is regulated pH=2.0~5.0) or 0.2%~5% glacial acetic acid or 0.2%~5% formic acid or 0.2%~3% phosphoric acid solution, gradient elution, flow velocity is that 0.5~2.0ml/min, detection wavelength are one or several in the 190-400nm scope, and column temperature is 20~60 ℃;
(4) based on the formulation of the standard finger-print of Herba Erigerontis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Herba Erigerontis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00;
B, adopt liquid chromatography test Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics be main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Ginseng Rubra or Radix Ginseng and the Radix Ophiopogonis medical material, comprise the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, ophiopogonin D ' in one or more, water or methanol, dissolve with ethanol are diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 20%~99% acetonitrile or 20%~99% methanol: 0.01mol/L~2mol/L sodium dihydrogen phosphate or 0.2%~3% glacial acetic acid or 0.2%~3% formic acid or 0.2%~3% phosphoric acid solution, gradient elution, flow velocity is that 0.5~2.0ml/min, detection wavelength are one or several in the 190-300nm scope, and column temperature is 20~60 ℃;
(4) based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the formulation of standard finger-print: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with said method as Radix Ginseng Rubra or Radix Ginseng in the injection to be measured and Radix Ophiopogonis ingredients fingerprint means of testing;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00;
C, employing liquid chromatography are tested the finger printing based on the Fructus Schisandrae Chinensis composition characteristics:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds water or methanol, dissolve with ethanol or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active product in contrast in an amount of schisandra chinensis medicinal material, comprise in schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schizantherin the second grade one or more, water or methanol, ethanol dilution are as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is acetonitrile or methanol: water or 0.01%~1% phosphoric acid solution or 0.2%~3% glacial acetic acid solution or 0.2%~3% formic acid solution, gradient elution, flow velocity are that 0.5~1.5ml/min, detection wavelength are 203~390nm, and column temperature is 20~50 ℃;
(4) based on the formulation of the standard finger-print of Fructus Schisandrae Chinensis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Fructus Schisandrae Chinensis ingredients fingerprint in the injection to be measured;
(6) with injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.80~1.00;
D, employing liquid chromatography test Radix Codonopsis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that scutellarin, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance in an amount of codonopsis pilosula, comprise in lobetyolin, the atractylenoide one or more, water or methanol, dissolve with ethanol are diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 5%~99%: 95%~5% acetonitrile or methanol-water or 0.01mol/L~2mol/L sodium dihydrogen phosphate or 0.2%~3% glacial acetic acid or 0.2%~3% formic acid or 0.2%~3% phosphoric acid solution, flow velocity is 0.5~2.0ml/min, detect wavelength is that one or several or evaporation photodetector in the 190-300nm scope detects, and column temperature is 20~60 ℃;
(4) based on the formulation of the standard finger-print of Radix Codonopsis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Radix Codonopsis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00.
Embodiment 2 finger printing
A, adopt the finger printing of liquid chromatography for measuring based on the Herba Erigerontis composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: it is an amount of that precision takes by weighing scutellarin, adds methanol and make the solution that every 1ml contains 0.1mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is an acetonitrile, Mobile phase B is 0.02moL/L sodium dihydrogen phosphate (25% phosphoric acid is transferred pH=3.5), gradient elution, solvent ratios was from 0 minute to 8 minutes, and the ratio of mobile phase A rises to 30% by 18%, from 8 minutes to 15 minutes, the ratio of acetonitrile rises to 50% by 30%, and from 15 minutes to 18 minutes, the ratio of acetonitrile reduced to 18% by 50%, from 18 minutes to 60 minutes, the ratio of acetonitrile was 18%; Flow velocity is 1.0ml/min, and the detection wavelength is 283 ± 2nm, and column temperature is 40 ℃;
(4) based on the formulation of the standard finger-print of Herba Erigerontis composition characteristics: accurate respectively each the 10 μ l of object of reference solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with the means of testing of said method as Herba Erigerontis ingredients fingerprint in the pulse restoring injection to be measured;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
B, adopt liquid chromatography for measuring based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg
1In right amount, add methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is the 0.05mol/L potassium dihydrogen phosphate, and Mobile phase B is acetonitrile-water (80: 20), gradient elution, solvent ratios was from 0 minute to 5 minutes, the ratio of Mobile phase B is 25%, and from 5 minutes to 40 minutes, the ratio of Mobile phase B rose to 64% by 25%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 64%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 80% by 64%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 25% by 80%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;
(4) based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the formulation of standard finger-print: accurate respectively each the 10 μ l of object of reference solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with said method as Radix Ginseng Rubra or Radix Ginseng in the pulse restoring injection to be measured and Radix Ophiopogonis ingredients fingerprint means of testing;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
C, adopt the finger printing of liquid chromatography for measuring based on the Fructus Schisandrae Chinensis composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the schisandrin reference substance, adds water and make the solution that every 1ml contains 0.1mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is methanol-water, gradient elution, solvent ratios are from 0 minute to 6 minutes, and the ratio of methanol is 68%, from 6 minutes to 8 minutes, the ratio of methanol rises to 76% by 68%, and from 8 minutes to 10 minutes, the ratio of methanol was 76%, from 10 minutes to 15 minutes, the ratio of methanol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methanol was 68%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;
(4) based on the formulation of the standard finger-print of Fructus Schisandrae Chinensis composition characteristics: accurate respectively each the 10 μ l of reference substance solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with the means of testing of said method as Fructus Schisandrae Chinensis ingredients fingerprint in the pulse restoring injection to be measured;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
D, employing liquid chromatography test Radix Codonopsis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that scutellarin, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active atractylenoide in an amount of codonopsis pilosula, be diluted to suitable concn, as object of reference solution with dissolve with methanol;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 67%: 33% methanol-water, and flow velocity is that 1.0ml/min, evaporation photodetector detect, 30 ℃ of drift tube temperatures, and carrier gas flux 2.2L/min, column temperature are 30 ℃;
(4) based on the formulation of the standard finger-print of Radix Codonopsis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Radix Codonopsis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.90~1.00.
Embodiment 3 differentiates
The thin layer chromatography discrimination method of Herba Erigerontis medical material, scutellarin in a, the injection:
It is an amount of to get each preparation, adds ethyl acetate or ethanol equal solvent and extracts, and filters, and filtrate volatilizes, and residue is with the dissolving of methanol equal solvent, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Herba Erigerontis control medicinal material, shines medical material solution in pairs with legal system; Get the scutellarin reference substance again, add the methanol equal solvent and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254Lamellae, with ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1~60: 0.2~10: 0.3~5 or benzene or toluene-ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1~30: 0.5~15: 0.05~5: 0.01~3 is developing solvent, launch, take out, dry, spray is with 0.3~10% aluminum chloride ethanol or 0.3~10% aluminum chloride methanol solution, 105 ℃ were dried by the fire 1~15 minute, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of scutellarin in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~95%: 95%~5% methanol or acetonitrile-water or 0.05%~10% glacial acetic acid aqueous solution or 0.02%~2% phosphate aqueous solution or 2%~30%: 2%~30%: 96%~40% methanol or acetonitrile-oxolane-0.02%~2% phosphate aqueous solution or acetonitrile-0.005~0.3moL/L sodium dihydrogen phosphate (1~99% phosphoric acid is transferred pH=2.0~5.0) gradient elution system is a mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
C. the thin layer chromatography discrimination method of schisandra chinensis medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the injection:
It is an amount of to get each preparation, adds chloroform or ether or ethyl acetate or normal hexane or 10~95% ethanol equal solvents and extracts, and filters, and filtrate volatilizes, and residue adds chloroform or the dissolving of ethyl acetate equal solvent, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Fructus Schisandrae Chinensis control medicinal material, shines medical material solution in pairs with legal system; Get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B reference substance again, add chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned ten kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254Lamellae, with petroleum ether (30~60 ℃) or petroleum ether (60~90 ℃)-Ethyl formate or ethyl acetate-formic acid or acetic acid 2~40: 0.2~15: 0.1~5 upper solution or benzene or toluene-ethyl acetate or Ethyl formate 1~30: 0.2~10 or normal hexane-benzene or toluene-Ethyl formate or ethyl acetate-formic acid or acetic acid 0.2~5: 0.5~10: 1~15: 0.1~5 are developing solvent, launch, take out, dry, putting uviol lamp 365nm or 254nm inspects or sprays with 2~20% chromotropic acids-concentrated sulfuric acid solution or 2~20% phosphomolybdic acid ethanol solutions or anisaldehyde sulfuric acid solution, 80 ℃~160 ℃ are dried by the fire the clear or smoked colour developing of iodine to the speckle colour developing, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
The liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in d, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
In e, the injection Radix Ophiopogonis medical material the thin layer chromatography discrimination method:
It is an amount of to get each preparation, adds the n-butyl alcohol equal solvent and extracts, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets control medicinal material Radix Ophiopogonis, adds chloroform-methanol or ethyl acetate or n-butyl alcohol equal solvent and extracts, and filters, and evaporate to dryness, residue add chloroform dissolving, medical material solution in contrast; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned three kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with benzene or toluene or chloroform-methanol or ethanol-glacial acetic acid or formic acid or water 8~300: 0.5~100: 0.01~30 or ethyl acetate or Ethyl formate-pyridine-water 0.2~10: 0.1~5: 0.2~10 is developing solvent, launch, take out, dry, put and inspect under uviol lamp 365nm or the 254nm or spray with 10% sulphuric acid or vanillin reagent or 50% sulphuric acid or 5% vanillin reagent or anisaldehyde reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, the fluorescence speckle that should show same color, negative noiseless;
Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:
It is an amount of to get each preparation, and the water equal solvent extracts back n-butyl alcohol and ether or the extraction of ethyl acetate equal solvent, and the extract evaporate to dryness is with the dissolving of methanol equal solvent, as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ', add the methanol equal solvent respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four liquors, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with ethyl acetate or chloroform-methanol-water 3~50: 1~15: 0.1~5 is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
Ophiopogonin B in g, the injection, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in h, the injection:
It is an amount of to get each preparation, adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, regulates pH to neutral, evaporate to dryness, and residue is with chloroform or the dissolving of ethyl acetate equal solvent, as need testing solution.Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, adds chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four liquid, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with normal hexane or methanol-ethyl acetate or chloroform or Ethyl formate-water 0.2~15: 0.2~40: 0.1~5 is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
The liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, take out, put, transfer pH, extract with the chloroform equal solvent to neutral to room temperature, the extracting solution evaporate to dryness, residue filters with microporous filter membrane with the dissolving of methanol equal solvent, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in j, the injection
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:
It is an amount of to get each preparation, extracts with n-butyl alcohol or ethanol equal solvent, filters, and filtrate is as need testing solution; Get Radix Ginseng Rubra or Radix Ginseng control medicinal material, add the chloroform reflux, extract,, discard chloroform solution, residue adds the water-saturated n-butanol equal solvent and extracts, and extracting solution adds ammonia solution, and divide and get the upper strata, evaporate to dryness, residue dissolves with the methanol equal solvent, in contrast medical material solution; Other gets the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add dissolvings such as methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned 7 kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, lower floor's solution or n-butyl alcohol-ethyl acetate or the Ethyl formate-water 1~10: 0.2~2 placed below 5~40: 10~100: 5~50: 0.2~3010 ℃ with chloroform-ethyl acetate or Ethyl formate-methanol-water: 1~15 upper strata is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent or 50% sulphate reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color, negative noiseless;
K. ginsenoside Rg in the injection
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds water or methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution for the contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels, 5%~40%: 95%~60% methanol or acetonitrile-water or 0.5%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution or 0.5%~5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5~2.0ml/min, the detection wavelength is in 200~350nm scope or evaporate the photodetector detection, and column temperature is in 20~50 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of lobetyolin in L, the injection:
It is an amount of to get each preparation, is dissolved in water, and with the extraction of n-butyl alcohol equal solvent, extract is concentrated into dried, use dissolve with methanol,, or put in C18 or the C8 solid phase extraction column, use 5%~50% methanol, methanol-eluted fractions successively as need testing solution, collect meoh eluate, be concentrated into 1ml, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Radix Codonopsis control medicinal material, shines medical material solution in pairs with legal system; Other gets lobetyolin's reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, n-butyl alcohol-glacial acetic acid or formic acid or ethanol or dehydrated alcohol-water 1~35: 0.1~10: 0.05~5 are developing solvent, launch, and take out, dry, spray is with 3%~30% ethanol solution of sulfuric acid, and 80~150 ℃ were heated 1~30 minute, and put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the fluorescence speckle of same color, negative noiseless;
The liquid chromatograph discrimination method of lobetyolin in m, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.
Embodiment 4 differentiates
The thin layer chromatography discrimination method of Herba Erigerontis medical material, scutellarin in a, the injection:
It is an amount of to get each preparation, adds ethyl acetate extraction, filters, and filtrate volatilizes, and the residue dissolve with methanol is as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Herba Erigerontis control medicinal material, shines medical material solution in pairs with legal system; Get the scutellarin reference substance again, add the methanol equal solvent and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate, toluene-ethyl acetate-formic acid-water 10: 5: 1: 0.5 is developing solvent, launch, take out, dry, spray is with 3% aluminum chloride alcoholic solution, 105 ℃ were dried by the fire 5 minutes, and put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the same color speckle;
The liquid chromatograph discrimination method of scutellarin in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.1% phosphate aqueous solution is a mobile phase at 20%: 80%, and the detection wavelength is 335nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
C. the thin layer chromatography discrimination method of Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the injection:
It is an amount of to get each preparation, adds chloroform and extracts, and filters, and filtrate volatilizes, and residue adds the chloroform dissolution with solvents, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Fructus Schisandrae Chinensis control medicinal material, shine medical material solution in pairs with legal system, get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B reference substance again, add chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned ten kinds of solution, put in same silica gel G F respectively
254Lamellae, with petroleum ether (30~60 ℃)-upper solution of 15: 5: 1 of Ethyl formate-formic acid is developing solvent, launch, take out, dry, put uviol lamp 254nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless;
The liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in d, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Radix Ophiopogonis in e, the injection:
It is an amount of to get each preparation, adds chloroform-methanol (7: 3) mixed solution and extracts, and filters, and evaporate to dryness, residue add the chloroform dissolving, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets control medicinal material Radix Ophiopogonis, adds chloroform-methanol and extracts, and filters, and evaporate to dryness, residue add chloroform dissolving, medical material solution in contrast; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned three kinds of solution, put in same silica gel G F respectively
254On the lamellae, be at 80: 5: 0.1 developing solvent, launch, inspect under the 254nm with toluene-methanol-glacial acetic acid, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color, negative noiseless;
Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:
It is an amount of to get each preparation, with using n-butanol extraction behind the water dissolution, and the extract evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ', add the methanol equal solvent respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate-methanol-water is developing solvent at 15: 5: 1, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that shows same color, negative noiseless;
Ophiopogonin B in g, the injection, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in h, the injection:
It is an amount of to get each preparation, adds 3% sulphuric acid hydrolysis 4 hours, regulates pH to neutral, evaporate to dryness, and residue dissolves with chloroform, as need testing solution.Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, adds chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four liquid, put respectively on same silica gel g thin-layer plate, with normal hexane-ethyl acetate or-water is developing solvent at 1: 1: 1, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 90 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that shows same color, negative noiseless;
The liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulphuric acid, takes out, and puts to room temperature, transfers pH to neutral, uses chloroform extraction, the extracting solution evaporate to dryness, and the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in j, the injection
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:
Get this product, use n-butanol extraction, filter, filtrate is as need testing solution; Get Radix Ginseng Rubra or Radix Ginseng control medicinal material, add the chloroform reflux, extract,, discard chloroform solution, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; Other gets the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add dissolvings such as methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned 7 kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: lower floor's solution of placing below 1010 ℃ was developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless;
K. ginsenoside Rg in the injection
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of lobetyolin in l, the injection:
It is an amount of to get each preparation, add methanol 25ml, supersound process 30 minutes filters, the filtrate evaporate to dryness, residue adds water 2ml makes dissolving, puts in the C18 solid phase extraction column (500mg is with methanol, each the 10ml prewashing of 20% methanol), use 20% methanol, each 5ml eluting of methanol successively, collect meoh eluate, be concentrated into 1ml, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Radix Codonopsis control medicinal material, shines medical material solution in pairs with legal system; It is an amount of that other gets lobetyolin's reference substance, adds methanol and make every 1ml and contain 1mg solution, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw need testing solution 6 μ l, reference substance solution 2 μ l, put respectively on same high-efficient silica gel G lamellae, with n-butyl alcohol-glacial acetic acid-water (7: 1: 0.5) is developing solvent, launch to take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ were heated 5 minutes, and 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, negative noiseless;
The liquid chromatograph discrimination method of lobetyolin in m, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.
Embodiment 5: assay
The assay of scutellarin in a, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~95%: 95%~5% methanol or acetonitrile-water or 0.05%~10% glacial acetic acid aqueous solution or 0.02%~2% phosphate aqueous solution or 2%~30%: 2%~30%: 96%~40% methanol or acetonitrile-oxolane-0.02%~2% phosphate aqueous solution or acetonitrile-0.005~0.3moL/L sodium dihydrogen phosphate (1~99% phosphoric acid is transferred pH=2.0~5.0) gradient elution system is a mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 6.0mg;
Content of total flavone is measured in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol and makes dissolving and fixed to scale in right amount, shakes up, and precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), measure trap at the wavelength place of 335 ± 10nm or get each preparation an amount of, put in the measuring bottle, thin up is to scale, shake up, precision is measured in right amount, puts in the 10ml measuring bottle, adds 50% methanol or water to 5ml, add 5% sodium nitrite solution, 0.3~1ml, shake up, placed 6 minutes, add 10% aluminum nitrate solution, 0.3~1ml, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4~10ml adds 50% methanol or water again to scale, shake up, as need testing solution; With rutin or scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at 500 ± 10nm place, calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the limit of total flavones in rutin or scutellarin, must not be less than 30.0mg;
C. ginsenoside Rg in the injection
1, ginsenoside Rb
1, ginsenoside Re's assay:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution for the contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels, 5%~40%: 95%~60% methanol or acetonitrile-water or 0.5%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution or 0.5%~5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5~2.0ml/min, the detection wavelength is in 200~350nm scope or evaporate the photodetector detection, and column temperature is in 20~50 ℃ of scopes; Calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.0mg, the limit that contains the ginsenoside Re must not be less than 1.5mg, contains ginsenoside Rb
1Limit must not be less than 1.0mg, contain the ginsenoside Rg
1, the ginsenoside Re the limit of summation must not be less than 2.5mg;
D. the assay of total saponins in the injection:
It is an amount of to get each preparation, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.2~4ml, perchloric acid 0.5~5ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, with ice-water bath cooling 2 minutes, precision added glacial acetic acid to 25ml immediately, shake up, as need testing solution; With the ginsenoside Rg
1Or ginsenoside Rb
1Or the product of ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at the wavelength place of 547 ± 10nm, calculates with external standard method or standard curve method, and each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg
1Or ginsenoside Rb
1Or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' meter, must not be less than 18.0mg;
E. total polysaccharides assay in the injection:
It is an amount of that monosaccharide is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg), convert out the content of every bottle of monosaccharide thus;
It is an amount of that total sugar is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 1~4 hour is put coldly, adds 1~2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents in every bottle in the sample with this;
The content of total polysaccharides during the content that deducts monosaccharide with sugar contents promptly gets every bottle;
Each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 70.0mg;
Total lignans assay in f, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use ethyl acetate extraction and extracting solution also, water bath method, and the residue dissolve with methanol is as need testing solution; With schisandrin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), wavelength place at 254 ± 10nm measures trap, calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 4.0mg;
Schisandrin, deoxyschizandrin, schisandrin B assay in g, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains schisandrin must not be less than 0.3mg, and the limit that contains deoxyschizandrin must not be less than 0.3mg; Contain schisandrin B (C
24H
32O
6) limit must not be less than 0.3mg; The limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.9mg;
Ophiopogonin B, ophiopogonin D, ophiopogonin D ' assay in h, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is that 200~410nm or evaporation photodetector detect 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains ophiopogonin B must not be less than 0.05mg, and the limit that contains ophiopogonin D must not be less than 0.05mg; Contain ophiopogonin D ' limit must not be less than 0.01mg; Contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.11mg;
Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin assay in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, take out, put, transfer pH, use chloroform extraction to neutral to room temperature, the extracting solution evaporate to dryness, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is that 200~410nm or evaporation photodetector detect 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains Ruscus aculeatus L. sapogenin must not be less than 0.01mg, and the limit that contains diosgenin must not be less than 0.01mg; The limit that contains ruscogenin must not be less than 0.01mg; The limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.03mg;
The assay of lobetyolin in j, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains lobetyolin must not be less than 0.1mg.
Embodiment 6: assay
The assay of scutellarin in a, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, acetonitrile-0.1% phosphate aqueous solution (20%: 80%) is a mobile phase, the detection wavelength is 335nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 6.0mg;
Content of total flavone is measured in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol and makes dissolving and fixed to scale in right amount, shakes up, and precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2000 A), wavelength place at 335nm measures trap, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains the limit of total flavones in scutellarin, must not be less than 30.0mg;
C. ginsenoside Rg in the injection
1, ginsenoside Rb
1, ginsenoside Re's assay:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is in 10~50 ℃ of scopes; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.0mg, the limit that contains the ginsenoside Re must not be less than 0.5mg, contains ginsenoside Rb
1Limit must not be less than 1.0mg, contain the ginsenoside Rg
1, the ginsenoside Re the limit of summation must not be less than 2.5mg;
D. the assay of total saponins in the injection:
It is an amount of to get each preparation, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method, take out immediately, precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml, shake up, heating is 15 minutes in 60 ℃ of water-baths, takes out, immediately with ice-water bath cooling 2 minutes, precision adds glacial acetic acid to 25ml, shaking up, is blank with the retinue solvent, according to spectrophotography, wavelength place at 547nm measures trap, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg
1Meter must not be less than 18.0mg;
E. total polysaccharides assay in the injection:
It is an amount of that monosaccharide is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg), convert out the content of every bottle of monosaccharide thus;
It is an amount of that total sugar is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 1~4 hour is put coldly, adds 1~2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents in every bottle in the sample with this;
The content of total polysaccharides during the content that deducts monosaccharide with sugar contents promptly gets every bottle;
Each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 70.0mg;
Total lignans assay in f, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use ethyl acetate extraction and and extracting solution, water bath method, the residue dissolve with methanol is a blank with the retinue solvent, according to spectrophotography, wavelength place at 254nm measures trap, calculates with one point external standard method, and each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 4.0mg;
Schisandrin, deoxyschizandrin, schisandrin B assay in g, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65: 35, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains schisandrin must not be less than 0.3mg, and the limit that contains deoxyschizandrin must not be less than 0.3mg; Contain schisandrin B (C
24H
32O
6) limit must not be less than 0.3mg; The limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.9mg;
Ophiopogonin B, ophiopogonin D, ophiopogonin D ' assay in h, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water (90: 10) is a mobile phase, detecting wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains ophiopogonin B must not be less than 0.05mg, and the limit that contains ophiopogonin D must not be less than 0.05mg; Contain ophiopogonin D ' limit must not be less than 0.01mg; Contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.11mg;
Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin assay in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulphuric acid, takes out, put to room temperature, transfer pH, use chloroform extraction and extracting solution also to neutral, evaporate to dryness, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains Ruscus aculeatus L. sapogenin must not be less than 0.01mg, and the limit that contains diosgenin must not be less than 0.01mg; The limit that contains ruscogenin must not be less than 0.01mg; The limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.03mg;
The assay of lobetyolin in j, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains lobetyolin must not be less than 0.1mg.
Claims (8)
1, a kind of method of quality control of the traditional medicine Injectio made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis, it is characterized in that: this method comprises following all or part of content:
(1) finger printing test, comprise based on the finger printing of Herba Erigerontis composition characteristics based on the finger printing of Radix Codonopsis composition characteristics and based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing and based on the finger printing of Fructus Schisandrae Chinensis composition characteristics;
(2) Herba Erigerontis medical material, Radix Ginseng Rubra or Radix Ginseng or codonopsis pilosula, Radix Ophiopogonis medical material, schisandra chinensis medicinal material, scutellarin, ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the differential test method of all or part of composition in Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B, lobetyolin, atractylenoide etc.;
(3) scutellarin, ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the content test method of all or part of composition such as Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schisandrin, schisantherin B, schisantherin A, deoxyschizandrin, schisandrin B, schisandrin C, total saponins, total lignans, total flavones, total polysaccharides, lobetyolin, atractylenoide.
2, according to the method for quality control of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis of claim 1, it is characterized in that this method comprises one or more in the following finger printing:
A, employing liquid chromatography test Herba Erigerontis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance scutellarin in an amount of Herba Erigerontis medical material, be diluted to suitable concn with methanol or dissolve with ethanol, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is acetonitrile or methanol: 0.005mol/L~2mol/L sodium dihydrogen phosphate (phosphoric acid is regulated pH=2.0~5.0) or 0.2%~5% glacial acetic acid or 0.2%~5% formic acid or 0.2%~3% phosphoric acid solution, gradient elution, flow velocity is that 0.5~2.0ml/min, detection wavelength are one or several in the 190-400nm scope, and column temperature is 20~60 ℃;
(4) based on the formulation of the standard finger-print of Herba Erigerontis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Herba Erigerontis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00;
B, adopt liquid chromatography test Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics be main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Ginseng Rubra or Radix Ginseng and the Radix Ophiopogonis medical material, comprise the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, ophiopogonin D ' in one or more, water or methanol, dissolve with ethanol are diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 20%~99% acetonitrile or 20%~99% methanol: 0.01mol/L~2mol/L sodium dihydrogen phosphate or 0.2%~3% glacial acetic acid or 0.2%~3% formic acid or 0.2%~3% phosphoric acid solution, gradient elution, flow velocity is that 0.5~2.0ml/min, detection wavelength are one or several in the 190-300nm scope, and column temperature is 20~60 ℃;
(4) based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the formulation of standard finger-print: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with said method as Radix Ginseng Rubra or Radix Ginseng in the injection to be measured and Radix Ophiopogonis ingredients fingerprint means of testing;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00;
C, employing liquid chromatography are tested the finger printing based on the Fructus Schisandrae Chinensis composition characteristics:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds water or methanol, dissolve with ethanol or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active product in contrast in an amount of schisandra chinensis medicinal material, comprise in schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schizantherin the second grade one or more, water or methanol, ethanol dilution are as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is acetonitrile or methanol: water or 0.01%~1% phosphoric acid solution or 0.2%~3% glacial acetic acid solution or 0.2%~3% formic acid solution, gradient elution, flow velocity are that 0.5~1.5ml/min, detection wavelength are 203~390nm, and column temperature is 20~50 ℃;
(4) based on the formulation of the standard finger-print of Fructus Schisandrae Chinensis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Fructus Schisandrae Chinensis ingredients fingerprint in the injection to be measured;
(6) with injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.80~1.00;
D, employing liquid chromatography test Radix Codonopsis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that scutellarin, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active reference substance in an amount of codonopsis pilosula, comprise in lobetyolin, the atractylenoide one or more, water or methanol, dissolve with ethanol are diluted to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is 5%~99%: 95%~5% acetonitrile or methanol-water or 0.01mol/L~2mol/L sodium dihydrogen phosphate or 0.2%~3% glacial acetic acid or 0.2%~3% formic acid or 0.2%~3% phosphoric acid solution, flow velocity is 0.5~2.0ml/min, detect wavelength is that one or several or evaporation photodetector in the 190-300nm scope detects, and column temperature is 20~60 ℃;
(4) based on the formulation of the standard finger-print of Radix Codonopsis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Radix Codonopsis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.80~1.00.
3, according to the method for quality control of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis of claim 2, it is characterized in that this method comprises one or more in the following finger printing:
A, adopt the finger printing of liquid chromatography for measuring based on the Herba Erigerontis composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: it is an amount of that precision takes by weighing scutellarin, adds methanol and make the solution that every 1ml contains 0.1mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is an acetonitrile, Mobile phase B is 0.02moL/L sodium dihydrogen phosphate (25% phosphoric acid is transferred pH=3.5), gradient elution, solvent ratios was from 0 minute to 8 minutes, and the ratio of mobile phase A rises to 30% by 18%, from 8 minutes to 15 minutes, the ratio of acetonitrile rises to 50% by 30%, and from 15 minutes to 18 minutes, the ratio of acetonitrile reduced to 18% by 50%, from 18 minutes to 60 minutes, the ratio of acetonitrile was 18%; Flow velocity is 1.0ml/min, and the detection wavelength is 283 ± 2nm, and column temperature is 40 ℃;
(4) based on the formulation of the standard finger-print of Herba Erigerontis composition characteristics: accurate respectively each the 10 μ l of object of reference solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with the means of testing of said method as Herba Erigerontis ingredients fingerprint in the pulse restoring injection to be measured;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
B, adopt liquid chromatography for measuring based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg
1In right amount, add methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is the 0.05mol/L potassium dihydrogen phosphate, and Mobile phase B is acetonitrile-water (80: 20), gradient elution, solvent ratios was from 0 minute to 5 minutes, the ratio of Mobile phase B is 25%, and from 5 minutes to 40 minutes, the ratio of Mobile phase B rose to 64% by 25%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 64%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 80% by 64%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 25% by 80%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;
(4) based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the formulation of standard finger-print: accurate respectively each the 10 μ l of object of reference solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with said method as Radix Ginseng Rubra or Radix Ginseng in the pulse restoring injection to be measured and Radix Ophiopogonis ingredients fingerprint means of testing;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
C, adopt the finger printing of liquid chromatography for measuring based on the Fructus Schisandrae Chinensis composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing this product, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the schisandrin reference substance, adds water and make the solution that every 1ml contains 0.1mg, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is methanol-water, gradient elution, solvent ratios are from 0 minute to 6 minutes, and the ratio of methanol is 68%, from 6 minutes to 8 minutes, the ratio of methanol rises to 76% by 68%, and from 8 minutes to 10 minutes, the ratio of methanol was 76%, from 10 minutes to 15 minutes, the ratio of methanol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methanol was 68%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;
(4) based on the formulation of the standard finger-print of Fructus Schisandrae Chinensis composition characteristics: accurate respectively each the 10 μ l of reference substance solution and need testing solution that draw, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, the formulation standard finger-print;
(5) with the means of testing of said method as Fructus Schisandrae Chinensis ingredients fingerprint in the pulse restoring injection to be measured;
(6) with pulse restoring injection finger printing to be measured and the contrast of above-mentioned standard finger-print, calculate similarity, should be 0.90~1.00;
D, employing liquid chromatography test Radix Codonopsis composition characteristics are main finger printing:
(1) preparation of need testing solution: it is an amount of to get the Chinese medicine that scutellarin, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis make, and adds dissolving of water or methanol equal solvent or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: get the main active atractylenoide in an amount of codonopsis pilosula, be diluted to suitable concn, as object of reference solution with dissolve with methanol;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase is the 67%:33% methanol-water, and flow velocity is that 1.0ml/min, evaporation photodetector detect, 30 ℃ of drift tube temperatures, and carrier gas flux 2.2L/min, column temperature are 30 ℃;
(4) based on the formulation of the standard finger-print of Radix Codonopsis composition characteristics: accurate draw need testing solution and object of reference solution an amount of, inject chromatograph of liquid respectively, according to measured collection of illustrative plates, formulate standard finger-print;
(5) with the means of testing of said method as Radix Codonopsis ingredients fingerprint in the injection to be measured;
(6) with the finger printing and the contrast of above-mentioned standard finger-print of injection to be measured, calculate similarity, should be 0.90~1.00.
4, according to the method for quality control of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis of claim 1, it is characterized in that: the discrimination method of described injection is following all or part of content:
The thin layer chromatography discrimination method of Herba Erigerontis medical material, scutellarin in a, the injection:
It is an amount of to get each preparation, adds ethyl acetate or ethanol equal solvent and extracts, and filters, and filtrate volatilizes, and residue is with the dissolving of methanol equal solvent, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Herba Erigerontis control medicinal material, shines medical material solution in pairs with legal system; Get the scutellarin reference substance again, add the methanol equal solvent and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254Lamellae, with ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1~60: 0.2~10: 0.3~5 or benzene or toluene-ethyl acetate or Ethyl formate-formic acid or acetic acid-water 1~30: 0.5~15: 0.05~5: 0.01~3 is developing solvent, launch, take out, dry, spray is with 0.3~10% aluminum chloride ethanol or 0.3~10% aluminum chloride methanol solution, 105 ℃ were dried by the fire 1~15 minute, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the same color speckle;
The liquid chromatograph discrimination method of scutellarin in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~95%: 95%~5% methanol or acetonitrile-water or 0.05%~10% glacial acetic acid aqueous solution or 0.02%~2% phosphate aqueous solution or 2%~30%: 2%~30%: 96%~40% methanol or acetonitrile-oxolane-0.02%~2% phosphate aqueous solution or acetonitrile-0.005~0.3moL/L sodium dihydrogen phosphate (1~99% phosphoric acid is transferred pH=2.0~5.0) gradient elution system is a mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
C. the thin layer chromatography discrimination method of schisandra chinensis medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the injection:
It is an amount of to get each preparation, adds chloroform or ether or ethyl acetate or normal hexane or 10~95% ethanol equal solvents and extracts, and filters, and filtrate volatilizes, and residue adds chloroform or the dissolving of ethyl acetate equal solvent, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Fructus Schisandrae Chinensis control medicinal material, shines medical material solution in pairs with legal system; Get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B reference substance again, add chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned ten kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254Lamellae, with petroleum ether (30~60 ℃) or petroleum ether (60~90 ℃)-Ethyl formate or ethyl acetate-formic acid or acetic acid 2~40: 0.2~15: 0.1~5 upper solution or benzene or toluene-ethyl acetate or Ethyl formate 1~30: 0.2~10 or normal hexane-benzene or toluene-Ethyl formate or ethyl acetate-formic acid or acetic acid 0.2~5: 0.5~10: 1~15: 0.1~5 are developing solvent, launch, take out, dry, putting uviol lamp 365nm or 254nm inspects or sprays with 2~20% chromotropic acids-concentrated sulfuric acid solution or 2~20% phosphomolybdic acid ethanol solutions or anisaldehyde sulfuric acid solution, 80 ℃~160 ℃ are dried by the fire the clear or smoked colour developing of iodine to the speckle colour developing, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
The liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in d, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
In e, the injection Radix Ophiopogonis medical material the thin layer chromatography discrimination method:
It is an amount of to get each preparation, adds the n-butyl alcohol equal solvent and extracts, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets control medicinal material Radix Ophiopogonis, adds chloroform-methanol or ethyl acetate or n-butyl alcohol equal solvent and extracts, and filters, and evaporate to dryness, residue add chloroform dissolving, medical material solution in contrast; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned three kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with benzene or toluene or chloroform-methanol or ethanol-glacial acetic acid or formic acid or water 8~300: 0.5~100: 0.01~30 or ethyl acetate or Ethyl formate-pyridine-water 0.2~10: 0.1~5: 0.2~10 is developing solvent, launch, take out, dry, put and inspect under uviol lamp 365nm or the 254nm or spray with 10% sulphuric acid or vanillin reagent or 50% sulphuric acid or 5% vanillin reagent or anisaldehyde reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, the fluorescence speckle that should show same color, negative noiseless;
Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:
It is an amount of to get each preparation, and the water equal solvent extracts back n-butyl alcohol and ether or the extraction of ethyl acetate equal solvent, and the extract evaporate to dryness is with the dissolving of methanol equal solvent, as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ', add the methanol equal solvent respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four liquors, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with ethyl acetate or chloroform-methanol-water 3~50: 1~15: 0.1~5 is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
Ophiopogonin B in g, the injection, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in h, the injection:
It is an amount of to get each preparation, adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, regulates pH to neutral, evaporate to dryness, and residue is with chloroform or the dissolving of ethyl acetate equal solvent, as need testing solution.Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, adds chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four liquid, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, with normal hexane or methanol-ethyl acetate or chloroform or Ethyl formate-water 0.2~15: 0.2~40: 0.1~5 is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that should show same color, negative noiseless;
The liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, take out, put, transfer pH, extract with the chloroform equal solvent to neutral to room temperature, the extracting solution evaporate to dryness, residue filters with microporous filter membrane with the dissolving of methanol equal solvent, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in j, the injection
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:
It is an amount of to get each preparation, extracts with n-butyl alcohol or ethanol equal solvent, filters, and filtrate is as need testing solution; Get Radix Ginseng Rubra or Radix Ginseng control medicinal material, add the chloroform reflux, extract,, discard chloroform solution, residue adds the water-saturated n-butanol equal solvent and extracts, and extracting solution adds ammonia solution, and divide and get the upper strata, evaporate to dryness, residue dissolves with the methanol equal solvent, in contrast medical material solution; Other gets the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add dissolvings such as methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned 7 kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F
254On the lamellae, lower floor's solution or n-butyl alcohol-ethyl acetate or the Ethyl formate-water 1~10: 0.2~2 placed below 5~40: 10~100: 5~50: 0.2~3010 ℃ with chloroform-ethyl acetate or Ethyl formate-methanol-water: 1~15 upper strata is developing solvent, launch, take out, dry, spray is with 5~50% sulphuric acid ethanol reagent or 50% sulphate reagent, 90 ℃~120 ℃ dry by the fire to speckle colour developing clear, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color, negative noiseless;
K. ginsenoside Rg in the injection
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds water or methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution for the contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels, 5%~40%: 95%~60% methanol or acetonitrile-water or 0.5%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution or 0.5%~5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5~2.0ml/min, the detection wavelength is in 200~350nm scope or evaporate the photodetector detection, and column temperature is in 20~50 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of lobetyolin in L, the injection:
It is an amount of to get each preparation, is dissolved in water, and with the extraction of n-butyl alcohol equal solvent, extract is concentrated into dried, use dissolve with methanol,, or put in C18 or the C8 solid phase extraction column, use 5%~50% methanol, methanol-eluted fractions successively as need testing solution, collect meoh eluate, be concentrated into 1ml, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Radix Codonopsis control medicinal material, shines medical material solution in pairs with legal system; Other gets lobetyolin's reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, n-butyl alcohol-glacial acetic acid or formic acid or ethanol or dehydrated alcohol-water 1~35: 0.1~10: 0.05~5 are developing solvent, launch, and take out, dry, spray is with 3%~30% ethanol solution of sulfuric acid, and 80~150 ℃ were heated 1~30 minute, and put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the fluorescence speckle of same color, negative noiseless;
The liquid chromatograph discrimination method of lobetyolin in m, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.
5, according to the method for quality control of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis of claim 4, it is characterized in that: the discrimination method of described injection is following one or more:
The thin layer chromatography discrimination method of Herba Erigerontis medical material, scutellarin in a, the injection:
It is an amount of to get each preparation, adds ethyl acetate extraction, filters, and filtrate volatilizes, and the residue dissolve with methanol is as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Herba Erigerontis control medicinal material, shines medical material solution in pairs with legal system; Get the scutellarin reference substance again, add the methanol equal solvent and make the solution that every 1ml contains 1mg; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four kinds of solution, put respectively in same silica gel g thin-layer plate, toluene-ethyl acetate-formic acid-water 10: 5: 1: 0.5 is developing solvent, launch, take out, dry, spray is with 3% aluminum chloride alcoholic solution, 105 ℃ were dried by the fire 5 minutes, and put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the same color speckle;
The liquid chromatograph discrimination method of scutellarin in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-0.1% phosphate aqueous solution is a mobile phase at 20%: 80%, and the detection wavelength is 335nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
C. the thin layer chromatography discrimination method of Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the injection:
It is an amount of to get each preparation, adds chloroform and extracts, and filters, and filtrate volatilizes, and residue adds the chloroform dissolution with solvents, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Fructus Schisandrae Chinensis control medicinal material, shine medical material solution in pairs with legal system, get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B reference substance again, add chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned ten kinds of solution, put in same silica gel G F respectively
254Lamellae, with petroleum ether (30~60 ℃)-upper solution of 15: 5: 1 of Ethyl formate-formic acid is developing solvent, launch, take out, dry, put uviol lamp 254nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless;
The liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in d, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Radix Ophiopogonis in e, the injection:
It is an amount of to get each preparation, adds chloroform-methanol (7: 3) mixed solution and extracts, and filters, and evaporate to dryness, residue add the chloroform dissolving, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets control medicinal material Radix Ophiopogonis, adds chloroform-methanol and extracts, and filters, and evaporate to dryness, residue add chloroform dissolving, medical material solution in contrast; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned three kinds of solution, put in same silica gel G F respectively
254On the lamellae, be at 80: 5: 0.1 developing solvent, launch, inspect under the 254nm with toluene-methanol-glacial acetic acid, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color, negative noiseless;
Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:
It is an amount of to get each preparation, with using n-butanol extraction behind the water dissolution, and the extract evaporate to dryness, the residue dissolve with methanol is as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ', add the methanol equal solvent respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate-methanol-water is developing solvent at 15: 5: 1, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that shows same color, negative noiseless;
Ophiopogonin B in g, the injection, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in h, the injection:
It is an amount of to get each preparation, adds 3% sulphuric acid hydrolysis 4 hours, regulates pH to neutral, evaporate to dryness, and residue dissolves with chloroform, as need testing solution.Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, adds chloroform respectively or the ethyl acetate equal solvent is made the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned four liquid, put respectively on same silica gel g thin-layer plate, with normal hexane-ethyl acetate or-water is developing solvent at 1: 1: 1, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 90 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the speckle that shows same color, negative noiseless;
The liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulphuric acid, takes out, and puts to room temperature, transfers pH to neutral, uses chloroform extraction, the extracting solution evaporate to dryness, and the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in j, the injection
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:
Get this product, use n-butanol extraction, filter, filtrate is as need testing solution; Get Radix Ginseng Rubra or Radix Ginseng control medicinal material, add the chloroform reflux, extract,, discard chloroform solution, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; Other gets the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add dissolvings such as methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1~30 μ l of above-mentioned 7 kinds of solution, put respectively on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water 15: 40: 22: lower floor's solution of placing below 1010 ℃ was developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, and 105 ℃ are dried by the fire to the speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color, negative noiseless;
K. ginsenoside Rg in the injection
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatograph differentiate:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless;
The thin layer chromatography discrimination method of lobetyolin in l, the injection:
It is an amount of to get each preparation, add methanol 25ml, supersound process 30 minutes filters, the filtrate evaporate to dryness, residue adds water 2ml makes dissolving, puts in the C18 solid phase extraction column (500mg is with methanol, each the 10ml prewashing of 20% methanol), use 20% methanol, each 5ml eluting of methanol successively, collect meoh eluate, be concentrated into 1ml, as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Other gets the Radix Codonopsis control medicinal material, shines medical material solution in pairs with legal system; It is an amount of that other gets lobetyolin's reference substance, adds methanol and make every 1ml and contain 1mg solution, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw need testing solution 6 μ l, reference substance solution 2 μ l, put respectively on same high-efficient silica gel G lamellae, with n-butyl alcohol-glacial acetic acid-water (7: 1: 0.5) is developing solvent, launch to take out, dry, spray is with 10% ethanol solution of sulfuric acid, 105 ℃ were heated 5 minutes, and 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, negative noiseless;
The liquid chromatograph discrimination method of lobetyolin in m, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end, negative noiseless.
6, according to the method for quality control of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis of claim 1, it is characterized in that: the method for testing of described injection content should comprise one or more in following:
The assay of scutellarin in a, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~95%: 95%~5% methanol or acetonitrile-water or 0.05%~10% glacial acetic acid aqueous solution or 0.02%~2% phosphate aqueous solution or 2%~30%: 2%~30%: 96%~40% methanol or acetonitrile-oxolane-0.02%~2% phosphate aqueous solution or acetonitrile-0.005~0.3moL/L sodium dihydrogen phosphate (1~99% phosphoric acid is transferred pH=2.0~5.0) gradient elution system is a mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 6.0mg;
Content of total flavone is measured in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol and makes dissolving and fixed to scale in right amount, shakes up, and precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (appendix VA of Chinese Pharmacopoeia version in 2000), measure trap at the wavelength place of 335 ± 10nm or get each preparation an amount of, put in the measuring bottle, thin up is to scale, shake up, precision is measured in right amount, puts in the 10ml measuring bottle, adds 50% methanol or water to 5ml, add 5% sodium nitrite solution, 0.3~1ml, shake up, placed 6 minutes, add 10% aluminum nitrate solution, 0.3~1ml, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4~10ml adds 50% methanol or water again to scale, shake up, as need testing solution; With rutin or scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at 500 ± 10nm place, calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the limit of total flavones in rutin or scutellarin, must not be less than 30.0mg;
C. ginsenoside Rg in the injection
1, ginsenoside Rb
1, ginsenoside Re's assay:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution for the contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels, 5%~40%: 95%~60% methanol or acetonitrile-water or 0.5%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution or 0.5%~5% formic acid solution are mobile phase, gradient elution, flow velocity is 0.5~2.0ml/min, the detection wavelength is in 200~350nm scope or evaporate the photodetector detection, and column temperature is in 20~50 ℃ of scopes; Calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.0mg, the limit that contains the ginsenoside Re must not be less than 1.5mg, contains ginsenoside Rb
1Limit must not be less than 1.0mg, contain the ginsenoside Rg
1, the ginsenoside Re the limit of summation must not be less than 2.5mg;
D. the assay of total saponins in the injection:
It is an amount of to get each preparation, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.2~4ml, perchloric acid 0.5~5ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, with ice-water bath cooling 2 minutes, precision added glacial acetic acid to 25ml immediately, shake up, as need testing solution; With the ginsenoside Rg
1Or ginsenoside Rb
1Or the product of ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography, measures trap at the wavelength place of 547 ± 10nm, calculates with external standard method or standard curve method, and each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg
1Or ginsenoside Rb
1Or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' meter, must not be less than 18.0mg;
E. total polysaccharides assay in the injection:
It is an amount of that monosaccharide is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg), convert out the content of every bottle of monosaccharide thus;
It is an amount of that total sugar is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 1~4 hour is put coldly, adds 1~2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents in every bottle in the sample with this;
The content of total polysaccharides during the content that deducts monosaccharide with sugar contents promptly gets every bottle;
Each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 70.0mg;
Total lignans assay in f, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use ethyl acetate extraction and extracting solution also, water bath method, and the residue dissolve with methanol is as need testing solution; With schisandrin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (appendix VA of Chinese Pharmacopoeia version in 2000), wavelength place at 254 ± 10nm measures trap, calculate with external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 4.0mg;
Schisandrin, deoxyschizandrin, schisandrin B assay in g, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains schisandrin must not be less than 0.3mg, and the limit that contains deoxyschizandrin must not be less than 0.3mg; Contain schisandrin B (C
24H
32O
6) limit must not be less than 0.3mg; The limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.9mg;
Ophiopogonin B, ophiopogonin D, ophiopogonin D ' assay in h, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 1%~99%: 99%~1% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is that 200~410nm or evaporation photodetector detect 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains ophiopogonin B must not be less than 0.05mg, and the limit that contains ophiopogonin D must not be less than 0.05mg; Contain ophiopogonin D ' limit must not be less than 0.01mg; Contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.11mg;
Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin assay in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and adds 0.5~38% sulphuric acid or hydrochloric acid hydrolysis, take out, put, transfer pH, use chloroform extraction to neutral to room temperature, the extracting solution evaporate to dryness, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, detecting wavelength is that 200~410nm or evaporation photodetector detect 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains Ruscus aculeatus L. sapogenin must not be less than 0.01mg, and the limit that contains diosgenin must not be less than 0.01mg; The limit that contains ruscogenin must not be less than 0.01mg; The limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.03mg;
The assay of lobetyolin in j, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, 5%~80%: 95%~20% methanol or acetonitrile-water or 0.05%~5% glacial acetic acid aqueous solution or 0.02%~0.5% phosphate aqueous solution are mobile phase, the detection wavelength is 200~410nm, 20~50 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains lobetyolin must not be less than 0.1mg.
7, according to the method for quality control of the described Chinese medicine made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng or Radix Codonopsis, Radix Ophiopogonis and Fructus Schisandrae Chinensis of claim 6, it is characterized in that: the method for testing of described injection content is following one or more methods:
The assay of scutellarin in a, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol or mobile phase equal solvent to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol or alcoholic solution with the scutellarin reference substance are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, acetonitrile-0.1% phosphate aqueous solution (20%: 80%) is a mobile phase, the detection wavelength is 335nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains scutellarin must not be less than 6.0mg;
Content of total flavone is measured in b, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol and makes dissolving and fixed to scale in right amount, shakes up, and precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, as need testing solution; With scutellarin product in contrast, get reference substance solution with legal system.With the retinue solvent is blank, according to spectrophotography (appendix VA of Chinese Pharmacopoeia version in 2000), wavelength place at 335nm measures trap, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains the limit of total flavones in scutellarin, must not be less than 30.0mg;
C. ginsenoside Rg in the injection
1, ginsenoside Rb
1, ginsenoside Re's assay:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is in 10~50 ℃ of scopes; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.0mg, the limit that contains the ginsenoside Re must not be less than 0.5mg, contains ginsenoside Rb
1Limit must not be less than 1.0mg, contain the ginsenoside Rg
1, the ginsenoside Re the limit of summation must not be less than 2.5mg;
D. the assay of total saponins in the injection:
It is an amount of to get each preparation, puts in the 10ml measuring bottle, and adding distil water makes dissolving and fixed to scale in right amount, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method, take out immediately, precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml, shake up, heating is 15 minutes in 60 ℃ of water-baths, takes out, immediately with ice-water bath cooling 2 minutes, precision adds glacial acetic acid to 25ml, shaking up, is blank with the retinue solvent, according to spectrophotography, wavelength place at 547nm measures trap, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg
1Meter must not be less than 18.0mg;
E. total polysaccharides assay in the injection:
It is an amount of that monosaccharide is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg), convert out the content of every bottle of monosaccharide thus;
It is an amount of that total sugar is got each preparation, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 1~4 hour is put coldly, adds 1~2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents in every bottle in the sample with this;
The content of total polysaccharides during the content that deducts monosaccharide with sugar contents promptly gets every bottle;
Each preparation is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 70.0mg;
Total lignans assay in f, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use ethyl acetate extraction and and extracting solution, water bath method, the residue dissolve with methanol is a blank with the retinue solvent, according to spectrophotography, wavelength place at 254nm measures trap, calculates with one point external standard method, and each preparation is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 4.0mg;
Schisandrin, deoxyschizandrin, schisandrin B assay in g, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65: 35, and the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains schisandrin must not be less than 0.3mg, and the limit that contains deoxyschizandrin must not be less than 0.3mg; Contain schisandrin B (C
24H
32O
6) limit must not be less than 0.3mg; The limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.9mg;
Ophiopogonin B, ophiopogonin D, ophiopogonin D ' assay in h, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water (90: 10) is a mobile phase, detecting wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains ophiopogonin B must not be less than 0.05mg, and the limit that contains ophiopogonin D must not be less than 0.05mg; Contain ophiopogonin D ' limit must not be less than 0.01mg; Contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.11mg;
Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin assay in i, the injection:
It is an amount of to get each preparation, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulphuric acid, takes out, put to room temperature, transfer pH, use chloroform extraction and extracting solution also to neutral, evaporate to dryness, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, detecting wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains Ruscus aculeatus L. sapogenin must not be less than 0.01mg, and the limit that contains diosgenin must not be less than 0.01mg; The limit that contains ruscogenin must not be less than 0.01mg; The limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.03mg;
The assay of lobetyolin in j, the injection:
It is an amount of to get each preparation, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Take by weighing other flavour of a drug and adjuvant in the prescription ratio, make negative control solution with method; Methanol solution with lobetyolin's reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 22: 78, and the detection wavelength is 267nm, 30 ℃ of column temperatures; Calculate with one point external standard method, each preparation is unit quantity to be equivalent to every day with output, and the limit that the per unit amount contains lobetyolin must not be less than 0.1mg.
8, according to the method for quality control of claim 6 or the 7 described Chinese medicines made from Herba Erigerontis, Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis, it is characterized in that: the assay result of described injection, calculate scutellarin, total flavones, ginsenoside Rg according to percentage by weight
1, ginsenoside Rb
1, ginsenoside Re, total saponins, total polysaccharides, total lignans, schisandrin, deoxyschizandrin, schisandrin B, ophiopogonin B, ophiopogonin D, ophiopogonin D ', in the Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, the total content of all or part of material of lobetyolin, atractylenoide accounts for deduction adjuvant and outer more than 25% of total solid of moisture.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200510090430 CN1911393A (en) | 2005-08-12 | 2005-08-12 | Injection contg. traditional Chinese medicine, and its quality control method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200510090430 CN1911393A (en) | 2005-08-12 | 2005-08-12 | Injection contg. traditional Chinese medicine, and its quality control method |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1911393A true CN1911393A (en) | 2007-02-14 |
Family
ID=37720557
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 200510090430 Pending CN1911393A (en) | 2005-08-12 | 2005-08-12 | Injection contg. traditional Chinese medicine, and its quality control method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1911393A (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103105371A (en) * | 2011-11-10 | 2013-05-15 | 长白山制药股份有限公司 | Quality detection method of pharmaceutical composition injection |
CN104062365A (en) * | 2013-03-19 | 2014-09-24 | 石家庄以岭药业股份有限公司 | Determination method of content of schisandrin in medicament |
CN104569275A (en) * | 2015-01-26 | 2015-04-29 | 天津市第一中心医院 | Method for simultaneously measuring concentrations of monomer components including ursolic acid, schizandrin a, schizandrin b and schizandrin c |
CN109470795A (en) * | 2018-12-27 | 2019-03-15 | 云南生物谷药业股份有限公司 | The detection method of content of scutellarin in Chinese medicine |
CN109613154A (en) * | 2018-12-18 | 2019-04-12 | 贵阳中医学院 | A kind of method for building up of the peaceful codonopsis pilosula HPLC characteristic spectrum of prestige |
CN110412178A (en) * | 2019-09-11 | 2019-11-05 | 广西壮族自治区药用植物园 | A kind of Shengmai San Contents of Main Components measuring method |
-
2005
- 2005-08-12 CN CN 200510090430 patent/CN1911393A/en active Pending
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103105371A (en) * | 2011-11-10 | 2013-05-15 | 长白山制药股份有限公司 | Quality detection method of pharmaceutical composition injection |
CN103105371B (en) * | 2011-11-10 | 2015-03-18 | 长白山制药股份有限公司 | Quality detection method of pharmaceutical composition injection |
CN104062365A (en) * | 2013-03-19 | 2014-09-24 | 石家庄以岭药业股份有限公司 | Determination method of content of schisandrin in medicament |
CN104569275A (en) * | 2015-01-26 | 2015-04-29 | 天津市第一中心医院 | Method for simultaneously measuring concentrations of monomer components including ursolic acid, schizandrin a, schizandrin b and schizandrin c |
CN104569275B (en) * | 2015-01-26 | 2017-03-08 | 天津市第一中心医院 | The method simultaneously measuring ursolic acid and Fructus Schisandrae Chinensis the first and second C primes monomer component concentration |
CN109613154A (en) * | 2018-12-18 | 2019-04-12 | 贵阳中医学院 | A kind of method for building up of the peaceful codonopsis pilosula HPLC characteristic spectrum of prestige |
CN109470795A (en) * | 2018-12-27 | 2019-03-15 | 云南生物谷药业股份有限公司 | The detection method of content of scutellarin in Chinese medicine |
CN110412178A (en) * | 2019-09-11 | 2019-11-05 | 广西壮族自治区药用植物园 | A kind of Shengmai San Contents of Main Components measuring method |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1836717A (en) | Quality controlling method for pulse restoring injection | |
CN1689636A (en) | Quality controlling method for traditional Chinese medicine injection made from radix salvia miltiorrhiza and safflower | |
CN1853673A (en) | Quality controlling method of double-Danshen injection | |
CN1911395A (en) | Method for controlling quality of injection contg. traditional Chinese medicine | |
CN1939461A (en) | Quality control of Chinese medicinal compound preparation | |
CN1876022A (en) | Pharmaceutical composition with red sage root and Erigeron breviscapus for treating cerebrovascular disease, its preparation process and quality control method | |
CN1911393A (en) | Injection contg. traditional Chinese medicine, and its quality control method | |
CN101036774A (en) | Quality control method of compound cantharidin oral preparations | |
CN1939433A (en) | Quality control of Chinese medicinal compound preparation | |
CN101062294A (en) | Method for detecting the quality of the medicine combination for treating cerebral apoplexy and vasculitis | |
CN1911394A (en) | Method for controlling quality of injection contg. traditional Chinese medicine | |
CN1785263A (en) | Quality control method of compound polygonium oriental preparation | |
CN1853674A (en) | Quality controlling method of Xingdan injection | |
CN1939472A (en) | Quality control of compound preparation for treating diabetes | |
CN1939371A (en) | Quality control of Chinese-medicinal compound preparation | |
CN1958003A (en) | Preparation for treating chronic nasosinusitis, preparation method and quality control method | |
CN1939391A (en) | Quality control of Chinese-medicine compound preparation | |
CN1600365A (en) | Method for controlling quality of tonic semifluid extract of ten ingredients | |
CN1876028A (en) | Pharmaceutical preparation for treating coronary heart disease, its preparation process and quality control method | |
CN1939368A (en) | Quality control of Chinese medicinal compound preparation | |
CN1911396A (en) | Method for controlling quality injection contg. traditional Chinese medicine | |
CN1939392A (en) | Quality control of Chinese medicine compound preparation | |
CN1919308A (en) | Quality controlling means of traditional medicine Injectio | |
CN1726962A (en) | Method for controlling quality of cudrania wood and preparation | |
CN1209107C (en) | Carbazolyl alkaloid anticarcinogen and its prepn |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |