CN100529753C - Quality controlling method for pulse restoring injection - Google Patents
Quality controlling method for pulse restoring injection Download PDFInfo
- Publication number
- CN100529753C CN100529753C CNB2005102007595A CN200510200759A CN100529753C CN 100529753 C CN100529753 C CN 100529753C CN B2005102007595 A CNB2005102007595 A CN B2005102007595A CN 200510200759 A CN200510200759 A CN 200510200759A CN 100529753 C CN100529753 C CN 100529753C
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- Prior art keywords
- solution
- ginsenoside
- injection
- measured
- 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.)
- Expired - Fee Related
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Plant Substances (AREA)
- Steroid Compounds (AREA)
Abstract
The quality control method for Shengmai injection includes fingerprint test, identification, content measurement and other items. The present invention obtains ideal fingerprint of red ginseng or ginseng, ophiopogon root and schisandra in certain conditions. The quality control method is effective on the Chinese medicine injection prepared with red ginseng or ginseng, ophiopogon root and schisandra and possesses high precision and high stability.
Description
Technical field
The present invention is a kind of detection method of pulse restoring injection, belongs to the field of detection technique, particularly a kind of detection method for traditional medicine Injectio.
Background technology
Pulse restoring injection is that raw material is made with genseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine, have supplementing qi and nourishing yin, answer the effect that arteries and veins takes off admittedly, be used for QIYINLIANGXU, the palpitaition that feeble pulse is desired to take off, the deficiency of vital energy, peripheral coldness and miocardial infarction, cardiogenic shock, infectious shock also are used for the treatment of diseases such as coronary disease and angina pectoris, weakness of the spleen and stomach, acute viral myocarditis, malignant tumour, severe pneumonia of infants, viral pneumonia, virus hepatitis, optic atrophy, diabetes, cervical spondylopathy, psoriasis, sudden deafness at present clinically; Be to change agent by traditional Shengmai San to form, it has overcome the slow shortcoming of conventional formulation onset, number of research projects has been done to it by many inventors and medicine enterprise, as: number of patent application is " 93110807.1 ", application, number of patent application that name is called " shengmai injection and preparation technology thereof " are called the application of " a kind of shengmai injection and preparation method thereof " for " 96117457.9 ", name; Applicant's Yu Wenyong had also once applied for to Patent Office of the People's Republic of China that name was called: " a kind of pharmaceutical composition for the treatment of cardiovascular and cerebrovascular disease and preparation method thereof ", number of patent application are 02153312.1 patent of invention, but pharmaceutical preparation must be on the basis that guarantees the constant product quality controllable safety, constantly more new development, in order better to control the quality of said preparation, guarantee the security of medication, better instruct and produce, make technology controlling and process rationally strict more, make consumer's energy full appreciation product quality, need this parenteral solution method for quality of research control; At present, in the related drugs preparation of formulation of ' Sheng Mai ', generally only with ginsenoside Re, Rb
1Be to detect index, but its quality of reactor product comprehensively at all not very reasonable with this quality that is used for controlling parenteral solution only; If control,, relatively be difficult to implement owing to do not have ready-made detection scheme, testing conditions or the like with other index.
Summary of the invention
The objective of the invention is to: a kind of detection method of pulse restoring injection is provided, and this method provides means, technical method of the index that detects, detection or the like to relevant production, testing agency; So that better control the quality of said preparation, guarantee the security of medication, can better instruct production, make controlling of production process rationally strict more, make consumer's energy full appreciation product quality.
The present invention constitutes like this:
The detection method of pulse restoring injection comprises following all or part of content:
(1) finger-print of pulse restoring injection test, comprise based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics finger-print and based in the finger-print of fruit of Chinese magnoliavine composition characteristics one or more;
(2) red ginseng or ginseng crude drug, the tuber of dwarf lilyturf medicinal material, schisandra chinensis medicinal material, 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 the Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B;
(3) ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the content test method of all or part of composition in the Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schizandrin, wuweizi alcohol B, Schisantherin C, schizandrin A, deoxyschizandrin, schisandrin C, total saponins, total lignan, total polysaccharides.
The detection method of described pulse restoring injection, this method comprises one or both in the following finger-print:
A, adopt liquid phase chromatography test red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics be main finger-print:
(1) preparation of need testing solution: it is an amount of to get Shengmai injection to be measured, adds water or methyl alcohol or 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 reference substance in an amount of red ginseng or genseng and the tuber of dwarf lilyturf medicinal 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 a kind of, water or methyl alcohol or dissolve with ethanol, be settled to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase A is 0.01mol/L～2mol/L biphosphate sodium water solution or 0.01mol/L～2mol/L potassium dihydrogen phosphate aqueous solution or 0.01mol/L～2mol/L sodium hydrogen phosphate aqueous solution or water or 0.1%～5% glacial acetic acid solution or 0.1%～5% formic acid solution or 0.02%～5% phosphoric acid solution; B is 10%～100% acetonitrile solution or 10%～absolute methanol solution, and gradient elution, flow velocity be 0.5～2.0ml/min, detect wavelength is that one or several or evaporation photodetector in 190～410nm scope detects, and column temperature is in 20～60 ℃ of scopes;
(4) formulation of standard finger-print: with said method as formulate based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～50;
(5) based on the described method in (1)～(3) as red ginseng or genseng in the injection to be measured and the tuber of dwarf lilyturf composition characteristics the means of testing of finger-print, the finger-print of preparation testing sample;
(6) with the finger-print of injection to be measured and the contrast of above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of injection to be measured, should be 0.80～1.00;
II. in the injection finger-print to be measured, non-total peak area must not surpass 10% of total peak area;
In the ratio that 30%～80% total peak area arranged in the injection finger-print III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than soil 50%;
B, employing liquid phase chromatography are tested the finger-print based on fruit of Chinese magnoliavine composition characteristics:
(1) preparation of need testing solution: it is an amount of to get Shengmai injection to be measured, adds water or methyl alcohol or 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 reference substance in an amount of schisandra chinensis medicinal material, comprise a kind of in schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B, water or methyl alcohol or dissolve with ethanol, be settled to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is acetonitrile or methyl alcohol, Mobile phase B is water or 0.01%～3% phosphoric acid solution or 0.1%～5% glacial acetic acid solution or 0.1%～5% formic acid solution, gradient elution, flow velocity is that 0.5～2.0ml/min, detection wavelength are one or several in the 190-410nm scope, and column temperature is in 20～60 ℃ of scopes;
(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of fruit of Chinese magnoliavine composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～50;
(5) based on the means of testing of the described method in (1)～(3) as the finger-print of fruit of Chinese magnoliavine composition characteristics in the injection to be measured, the finger-print of preparation testing sample;
(6) with the contrast of injection finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of injection to be measured, should be 0.80～1.00;
II. in the injection finger-print to be measured, non-total peak area must not surpass 10% of total peak area;
In the ratio that 30%～80% total peak area arranged in the injection finger-print III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than ± 50%.
(ratio of total peak area is meant: as 1, calculate the ratio of each total fingerprint peaks area and object of reference peak area with the object of reference peak area)
The detection method of described pulse restoring injection, this method comprises one or both in the following finger-print:
A, adopt liquid chromatography for measuring based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics finger-print:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing Shengmai injection to be measured, adds water and make the solution that every 1ml contains 50mg, with the miillpore filter 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 methyl alcohol 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 is 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) formulation of standard finger-print: with said method as formulate based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;
(5) based on the described method in (1)～(3) as red ginseng or genseng in the injection to be measured and the tuber of dwarf lilyturf composition characteristics the means of testing of finger-print, the finger-print of preparation testing sample;
(6) with the contrast of pulse restoring injection finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of injection to be measured, should be 0.90～1.00;
II. in the injection finger-print to be measured, non-total peak area must not surpass 5% of total peak area;
In the ratio that 40%～60% total peak area arranged in the injection finger-print III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than ± 30%;
B, adopt the finger-print of liquid chromatography for measuring based on fruit of Chinese magnoliavine composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing Shengmai injection to be measured, adds water and make the solution that every 1ml contains 50mg, with the miillpore filter 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 schizandrin 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; Moving phase is methanol-water, gradient elution, solvent ratios are from 0 minute to 6 minutes, and the ratio of methyl alcohol is 68%, from 6 minutes to 8 minutes, the ratio of methyl alcohol rises to 76% by 68%, and from 8 minutes to 10 minutes, the ratio of methyl alcohol was 76%, from 10 minutes to 15 minutes, the ratio of methyl alcohol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methyl alcohol was 68%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;
(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of fruit of Chinese magnoliavine composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30
(5) based on the means of testing of the described method in (1)～(3) as the finger-print of fruit of Chinese magnoliavine composition characteristics in the injection to be measured, the finger-print of preparation testing sample;
(6) with the contrast of pulse restoring injection finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of injection to be measured, should be 0.90～1.00;
II. in the injection finger-print to be measured, non-total peak area must not surpass 5% of total peak area;
In the ratio that 40%～60% total peak area arranged in the injection finger-print III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than ± 30%.
The detection method of described pulse restoring injection, the discrimination method of described injection comprise following all or part of content:
One or more thin-layer chromatography discrimination method in the fruit of Chinese magnoliavine, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B in a, the injection:
It is an amount of to get Shengmai injection to be measured, adds methenyl choloride or ether or ethyl acetate or normal hexane or 10%～absolute ethyl alcohol or 10%～absolute methanol and extracts, and filters, and filtrate volatilizes, and residue adds methenyl choloride or ethyl acetate dissolving, as need testing solution; Other gets one or more preparation contrast solutions in fruit of Chinese magnoliavine control medicinal material, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B; The preparation of fruit of Chinese magnoliavine control medicinal material solution: it is an amount of to get fruit of Chinese magnoliavine control medicinal material, add methenyl choloride or ether or ethyl acetate or normal hexane or 10%～absolute ethyl alcohol or 10%～absolute methanol and extract, filter, filtrate volatilizes, residue adds methenyl choloride or ethyl acetate dissolving, medicinal material solution in contrast; The preparation of reference substance solution: get one or more reference substances in schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B; Add methenyl choloride or ethyl acetate respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned need testing solution and reference substance solution, put respectively in same silica gel g thin-layer plate or silica gel H thin layer plate or silica G F
254On the thin layer plate, with sherwood oil (30～60 ℃) or sherwood oil (60～90 ℃)-ethyl formate or ethyl acetate-formic acid or acetate 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 acetate 0.2～5: 0.5～10: 1～15: 0.1～5 are developping agent, launch, take out, dry, putting uviol lamp 365nm or 254nm inspects or sprays with 2%～20% chromotropic acid-concentrated sulfuric acid solution or 2%～20% phosphomolybdic acid ethanol solution or anisaldehyde sulfuric acid solution, dry by the fire the clear or smoked colour developing of iodine at 80 ℃～160 ℃ to the spot colour developing, in the test sample chromatogram, with control medicinal material chromatogram or the corresponding position of reference substance chromatogram on, should show the spot of same color;
One or more liquid chromatography discriminating in schizandrin, schizandrin A, the deoxyschizandrin in b, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methyl alcohol or moving phase or ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methyl alcohol or ethanolic solution with one or more reference substances in schizandrin, schizandrin A, the deoxyschizandrin are contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid 5%～80%: 95%～20% are moving phase, the detection wavelength is one or several in 190～410nm scope, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatogram, answer tool and the main peak in the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
The thin-layer chromatography discrimination method of the tuber of dwarf lilyturf in c, the injection:
It is an amount of to get Shengmai injection to be measured, adds methenyl choloride-methyl alcohol mixed solution or normal butyl alcohol or methenyl choloride or ethyl acetate or methylene chloride and extracts, as need testing solution; Other gets the control medicinal material tuber of dwarf lilyturf, adds methenyl choloride-methyl alcohol mixed solution or ethyl acetate or normal butyl alcohol or methenyl choloride or methylene chloride and extracts, and filters, and evaporate to dryness, residue add methenyl choloride or methylene chloride dissolving, medicinal material solution in contrast; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H thin layer plate or silica G F
254On the thin layer plate, with benzene or toluene or methenyl choloride or methylene chloride-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 developping agent, launch, take out, dry, put and inspect under uviol lamp 365nm or the 254nm or spray with 1%～50% sulfuric acid or vanillic aldehyde reagent or 1%～25% vanillin reagent or anisaldehyde reagent, it is clear to dry by the fire to the spot colour developing at 80 ℃～160 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, should show the fluorescence spot of same color;
Ophiopogonin B in d, the injection, ophiopogonin D, ophiopogonin D ' in one or more thin-layer chromatography discrimination method:
It is an amount of to get Shengmai injection to be measured, and with normal butyl alcohol or ether or ethyl acetate or methenyl choloride or dichloromethane extraction, extract volatilizes after the water dissolving, and residue is with methyl alcohol or dissolve with ethanol, as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ' in one or more reference substances, add methyl alcohol or ethanol respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H thin layer plate or silica G F
254On the thin layer plate, with ethyl acetate or methenyl choloride or methylene chloride-methanol or alcohol-water 3～50: 1～15: 0.1～5 is developping agent, launch, take out, dry, spray is with 5%～50% sulfuric acid ethanol reagent, it is clear to dry by the fire to the spot colour developing at 80 ℃～160 ℃, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, should show the spot of same color;
Ophiopogonin B in e, the injection, ophiopogonin D, ophiopogonin D ' in one or more liquid chromatography differentiate:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methyl alcohol or moving phase or ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methyl alcohol of one or more reference substances or ethanolic solution be contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid 1%～99%: 99%～1% are moving phase, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatogram, answer tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
One or more thin-layer chromatography discrimination method in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in f, the injection:
It is an amount of to get Shengmai injection to be measured, add to regulate pH behind sulfuric acid or the hydrochloric acid hydrolysis to neutral, and evaporate to dryness, residue is with methenyl choloride or methylene chloride or ethyl acetate or methyl alcohol or dissolve with ethanol, as need testing solution; Other gets one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin, adds methenyl choloride or methylene chloride or ethyl acetate or methyl alcohol or ethanol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ 1 of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H thin layer plate or silica G F
254On the thin layer plate, with normal hexane or cyclohexane or methyl alcohol or ethanol-ethyl acetate or methenyl choloride or methylene chloride or ethyl formate-water 0.2～15: 0.2～40: 0.1～5 is developping agent, launch, take out, dry, spray is with 5%～50% sulfuric acid ethanol reagent, it is clear to dry by the fire to the spot colour developing at 80 ℃～160 ℃, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, should show the spot of same color;
One or more liquid chromatography discriminating in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in g, the injection:
It is an amount of to get Shengmai injection to be measured, put in the round-bottomed flask, after vulcanization acid or hydrochloric acid hydrolysis are dissolved in water, take out, put to room temperature, with methenyl choloride or methylene chloride or normal butyl alcohol or ethyl acetate extraction, extract volatilizes accent pH to neutral back, residue filters with miillpore filter after with methyl alcohol or dissolve with ethanol, gets subsequent filtrate as need testing solution; With Ruscus aculeatus L. sapogenin, diosgenin, the methyl alcohol of one or more reference substances or ethanolic solution are contrast in the ruscogenin, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid 5%～95%: 95%～5% are moving phase, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatogram, answer tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Red ginseng or genseng, ginsenoside Rg in h, the injection
1, ginsenoside Rb
1, one or more thin-layer chromatography discrimination method among the ginsenoside Re, ginsenoside Rf:
It is an amount of to get Shengmai injection to be measured, with normal butyl alcohol or ethanol or methyl alcohol or ethyl acetate or methenyl choloride or methylene chloride extraction, filters, and filtrate is as need testing solution; Other gets red ginseng or genseng control medicinal material, ginsenoside Rg
1, ginsenoside Rb
1, among the ginsenoside Re, ginsenoside Rf one or more, the preparation contrast solution; The preparation of red ginseng or genseng control medicinal material solution: get red ginseng or the genseng control medicinal material is an amount of, adding methenyl choloride or methylene chloride reflux extracts, discard methenyl choloride or dichloromethane solution, residue adds water-saturated n-butanol or ethyl acetate extracts, extract adds ammonia solution, divides and gets upper strata, evaporate to dryness, residue is with methyl alcohol or dissolve with ethanol, medicinal material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg
1, ginsenoside Rb
1, one or more reference substances among the ginsenoside Re, ginsenoside Rf, add methyl alcohol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H thin layer plate or silica G F
254On the thin layer plate, lower floor's solution or normal 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 methenyl choloride or methylene chloride or normal hexane or cyclohexane-ethyl acetate or ethyl formate-methyl alcohol or ethanol or acetone-water: 1～15 upper strata is a developping agent, launch, take out, dry, spray is with 5%～50% sulfuric acid ethanol reagent, it is clear to dry by the fire to the spot colour developing at 80 ℃～160 ℃, put respectively under daylight and the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, should show the spot of same color;
Ginsenoside Rg in i, the injection
1, ginsenoside Rb
1, one or more liquid chromatography is differentiated among the ginsenoside Re:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methyl alcohol or moving phase or ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1The methyl alcohol of one or more reference substances or ethanolic solution are contrast among the ginsenoside Re, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% formic acid solution 5%～40%: 95%～60% are moving phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatogram, answer tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.
The detection method of described pulse restoring injection, the discrimination method of described injection comprise following all or part of content:
One or more thin-layer chromatography discrimination method in the fruit of Chinese magnoliavine, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B in a, the injection:
It is an amount of to get Shengmai injection to be measured, adds methenyl choloride and extracts, and filters, and filtrate volatilizes, and residue adds the methenyl choloride dissolving, as need testing solution; Other gets in fruit of Chinese magnoliavine control medicinal material, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B one or more, the preparation contrast solution; The preparation of fruit of Chinese magnoliavine control medicinal material solution: it is an amount of to get fruit of Chinese magnoliavine control medicinal material, adds methenyl choloride and extracts, and filters, and filtrate volatilizes, and residue adds methenyl choloride dissolving, medicinal material solution in contrast; The preparation of reference substance solution: get one or more reference substances in schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B, add methenyl choloride respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be developping agent, launch with sherwood oil (30～60 ℃)-upper solution of 15: 5: 1 of ethyl formate-formic acid, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
One or more liquid chromatography discriminating in schizandrin, schizandrin A, the deoxyschizandrin in b, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with one or more reference substances in schizandrin, schizandrin A, the deoxyschizandrin is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methanol-water is a moving phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
The thin-layer chromatography discrimination method of the tuber of dwarf lilyturf in c, the injection:
It is an amount of to get Shengmai injection to be measured, adds 7: 3 mixed solutions of methenyl choloride-methyl alcohol and extracts, and filters, and filtrate volatilizes, and residue adds the methenyl choloride dissolving, as need testing solution; Other gets the control medicinal material tuber of dwarf lilyturf, shines medicinal material solution in pairs with legal system; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be at 80: 5: 0.1 developping agent, launch, take out, dry, put under the uviol lamp 254nm and inspect with toluene-methyl alcohol-glacial acetic acid, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color, negative noiseless;
Ophiopogonin B in d, the injection, ophiopogonin D, ophiopogonin D ' in one or more thin-layer chromatography discrimination method:
It is an amount of to get Shengmai injection to be measured, and extracting n-butyl alcohol is used in water dissolving back, and extract volatilizes, and the residue dissolve with methanol is as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ' in one or more reference substances, add methyl alcohol respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developping agent with ethyl acetate-methanol-water at 15: 5: 1, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ dry by the fire to spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Ophiopogonin B in e, the injection, ophiopogonin D, ophiopogonin D ' in one or more liquid chromatography differentiate:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methanol solution of one or more reference substances be contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-water is a moving phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
One or more thin-layer chromatography discrimination method in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in f, the injection:
It is an amount of to get Shengmai injection to be measured, adds 3% sulphuric acid hydrolysis 4 hours, takes out, and puts to room temperature, regulates pH to neutral, filters, and filtrate volatilizes, and residue dissolves with methenyl choloride, as need testing solution; Other gets one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin, adds methenyl choloride respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developping agent with normal hexane-ethyl acetate-water at 1: 1: 1, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 90 ℃ dry by the fire to spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
One or more liquid chromatography discriminating in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in g, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the round-bottomed flask, and refluxing 4 hours with 3% sulfuric acid in the back that is dissolved in water, takes out, put to room temperature, transfer pH to neutral, extract with methenyl choloride, extract volatilizes, the residue dissolve with methanol filters with miillpore filter, gets subsequent filtrate as need testing solution; By the methanol solution with one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-water is a moving phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Red ginseng or genseng, ginsenoside Rg in h, the injection
1, ginsenoside Rb
1, one or more thin-layer chromatography discrimination method among the ginsenoside Re, ginsenoside Rf:
It is an amount of to get Shengmai injection to be measured, extracts with normal butyl alcohol, filters, and filtrate is as need testing solution; Other gets red ginseng or genseng control medicinal material, ginsenoside Rg
1, ginsenoside Rb
1, among the ginsenoside Re, ginsenoside Rf one or more, preparation control medicinal material solution; The preparation of red ginseng or genseng control medicinal material solution: get red ginseng or the genseng control medicinal material is an amount of, add the methenyl choloride refluxing extraction, discard methenyl choloride liquid, residue adds water-saturated n-butanol and extracts, and extract adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medicinal material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg
1, ginsenoside Rb
1, one or more reference substances among the ginsenoside Re, ginsenoside Rf, add methyl alcohol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methanol-water 15: 40: 22: 10 was developping agent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ are dried by the fire to spot colour developing clearly, put respectively under daylight and the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
Ginsenoside Rg in i, the injection
1, ginsenoside Rb
1, one or more liquid chromatography is differentiated among the ginsenoside Re:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1, the methanol solution of one or more reference substances is contrast among the ginsenoside Re, adopts liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, acetonitrile-water is a moving 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 chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.
The detection method of described pulse restoring injection, the method for testing of described injection content should comprise following all or part of content:
Ginsenoside Rg in a, the injection
1, ginsenoside Rb
1, one or more assay among the ginsenoside Re:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methyl alcohol or moving phase or ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1The methyl alcohol of one or more reference substances or ethanolic solution are contrast among the ginsenoside Re, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% formic acid solution 5%～40%: 95%～60% are moving phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with external standard method or calibration curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:
(1) the per unit amount contains the ginsenoside Rg
1Limit must not be less than 0.8mg;
(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.4mg;
(3) the per unit amount contains ginsenoside Rb
1Limit must not be less than 0.5mg;
(4) the per unit amount contains the ginsenoside Rg
1, the ginsenoside Re the limit of summation must not be less than 1.7mg;
The assay of total saponins in b, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, and adding distil water or methyl alcohol or ethanol make dissolving and fixed to scale in right amount, shake up, precision is measured in right amount, puts in the measuring bottle, water bath method takes out immediately, and precision adds 1%～50% vanillic aldehyde-glacial acetic acid solution 0.1ml～10ml, perchloric acid 0.1ml～15ml shakes up, and heats 3～50 minutes in 30 ℃～80 ℃ water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with the ginsenoside Rg
1Or ginsenoside Rb
1Or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures absorbance log, calculate with external standard method or calibration curve method, Shengmai injection to be measured 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 10mg;
Total polysaccharides assay in c, the injection:
It is an amount of that monose is got Shengmai injection to be measured, puts in the iodine flask, and pH is regulated to neutral with sodium hydroxide test solution in adding distil water dissolving back, 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, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate vs immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank test, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg), calculate the content of monose thus;
It is an amount of that total reducing sugar is got Shengmai injection to be measured, put in the iodine flask, adding distil water adds dilute sulfuric acid 25ml after making dissolving, reflux 1～4 hour, put cold, add 1～2 of instructions phenolphthalein solution, 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 vs immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank test, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg, calculates sugar contents with this;
The content that deducts monose with sugar contents promptly gets the content of total polysaccharides;
Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous dextrose, must not be less than 50mg;
Total lignan assay in d, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, and it is fixed to scale to add water, add hydrochloric acid precipitation saponin component after, with ethyl acetate or methenyl choloride or methylene chloride extracts and and extract, volatilize, residue is with methyl alcohol or dissolve with ethanol, as need testing solution; Methyl alcohol or ethanolic solution with schizandrin or wuweizi alcohol B are reference substance solution.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, wavelength place at 254 ± 10nm measures absorbance log, calculate with external standard method or calibration curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignan in schizandrin or wuweizi alcohol B, must not be less than 1.5mg;
One or more assay in schizandrin, schizandrin A, the deoxyschizandrin in e, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methyl alcohol or moving phase or ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methyl alcohol or ethanolic solution with one or more reference substances in schizandrin, schizandrin A, the deoxyschizandrin are contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid 5%～80%: 95%～20% are moving phase, the detection wavelength is one or several in 190～410nm scope, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or calibration curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:
(1) the per unit amount limit that contains schizandrin must not be less than 0.2mg;
(2) the per unit amount limit that contains schizandrin A must not be less than 0.025mg;
(3) the per unit amount limit that contains deoxyschizandrin must not be less than 0.075mg;
(4) the per unit amount limit that contains the summation of schizandrin, schizandrin A, deoxyschizandrin must not be less than 0.3mg;
Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' in one or more assay:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methyl alcohol or moving phase or ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methyl alcohol of one or more reference substances or ethanolic solution be contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid 1%～99%: 99%～1% are moving phase, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or calibration curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several.
(1) the per unit amount limit that contains ophiopogonin B must not be less than 0.05mg;
(2) the per unit amount limit that contains ophiopogonin D must not be less than 0.05mg;
(3) the per unit amount contain ophiopogonin D ' limit must not be less than 0.01mg;
(4) the per unit amount contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.11mg;
One or more assay in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in g, the injection:
It is an amount of to get Shengmai injection to be measured, put in the round-bottomed flask, after vulcanization acid or hydrochloric acid hydrolysis are dissolved in water, take out, put, transfer pH to extract with methenyl choloride or normal butyl alcohol or ethyl acetate to neutral back to room temperature, extract volatilizes, residue filters with miillpore filter with methyl alcohol or dissolve with ethanol, gets subsequent filtrate as need testing solution; With Ruscus aculeatus L. sapogenin, diosgenin, the methyl alcohol of one or more reference substances or ethanolic solution are contrast in the ruscogenin, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid 5%～95%: 95%～5% are moving phase, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or calibration curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:
(1) the per unit amount limit that contains Ruscus aculeatus L. sapogenin must not be less than 0.01mg;
(2) the per unit amount limit that contains diosgenin must not be less than 0.01mg;
(3) the per unit amount limit that contains ruscogenin must not be less than 0.01mg;
(4) the per unit amount limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.03mg.
The detection method of described pulse restoring injection, the method for testing of described injection content should comprise following all or part of content:
Ginsenoside Rg in a, the injection
1, ginsenoside Rb
1, one or more assay among the ginsenoside Re:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1, the methanol solution of one or more reference substances is contrast among the ginsenoside Re, adopts liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, acetonitrile-water is a moving 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 30 ℃; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:
(1) the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.6mg;
(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.8mg;
(3) the per unit amount contains ginsenoside Rb
1Limit must not be less than 1.0mg;
(4) the per unit amount contains the ginsenoside Rg
1, ginsenoside Rb
1, the ginsenoside Re the limit of summation must not be less than 3.4mg;
The assay of total saponins in b, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the 10ml measuring bottle, adds water to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillic aldehyde-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, immediately with ice-water bath cooling 2 minutes, fixed to scale with glacial acetic acid, shake up, as need testing solution; With the ginsenoside Rg
1Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, measure absorbance log, calculate with one point external standard method at the wavelength place of 547nm, Shengmai injection to be measured 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 20mg;
Total polysaccharides assay in c, the injection:
It is an amount of that monose is got Shengmai injection to be measured, 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg), calculate the content of monose thus;
It is an amount of that total reducing sugar is got Shengmai injection to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 4 hours 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg, calculates sugar contents with this;
The content that deducts monose with sugar contents promptly gets the content of total polysaccharides;
Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous dextrose, must not be less than 100mg;
Total lignan assay in d, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, and it is fixed to scale to add water, add hydrochloric acid precipitation saponin component after, with ethyl acetate extract and and extract, water bath method, the residue dissolve with methanol is as need testing solution; With the schizandrin is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, wavelength place at 254nm measures absorbance log, calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignan in schizandrin, must not be less than 3mg;
One or more assay in schizandrin, schizandrin A, the deoxyschizandrin in e, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with one or more reference substances in schizandrin, schizandrin A, the deoxyschizandrin is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methanol-water is a moving phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:
(1) the per unit amount limit that contains schizandrin must not be less than 0.4mg;
(2) the per unit amount limit that contains schizandrin A must not be less than 0.05mg;
(3) the per unit amount limit that contains deoxyschizandrin must not be less than 0.10mg;
(4) the per unit amount limit that contains the summation of schizandrin, schizandrin A, deoxyschizandrin must not be less than 0.55mg;
Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' in one or more assay:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methanol solution of one or more reference substances be contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-water is a moving phase at 90%: 10%, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:
(1) the per unit amount limit that contains ophiopogonin B must not be less than 0.1mg;
(2) the per unit amount limit that contains ophiopogonin D must not be less than 0.1mg;
(3) the per unit amount contain ophiopogonin D ' limit must not be less than 0.02mg;
(4) the per unit amount contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.22mg;
One or more assay in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in g, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulfuric acid, takes out, put to room temperature, transfer pH to neutral, with methenyl choloride extract and and extract, evaporate to dryness, the residue dissolve with methanol filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-water is a moving phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:
(1) the per unit amount limit that contains Ruscus aculeatus L. sapogenin must not be less than 0.02mg;
(2) the per unit amount limit that contains diosgenin must not be less than 0.02mg;
(3) the per unit amount limit that contains ruscogenin must not be less than 0.02mg;
(4) the per unit amount limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.06mg.
The detection method of described pulse restoring injection, all or part of total content of surveying composition of the saponins in the described injection, polysaccharide, lignanoids account for more than 25% of total solid of deducting auxiliary material amount and amount of moisture in the preparation.
(total solid is meant: the weight of content deducts the weight behind auxiliary material amount and the amount of moisture)
Compared with prior art, the present invention's quality of the Chinese medicine injection products made with red ginseng or genseng, the tuber of dwarf lilyturf and the fruit of Chinese magnoliavine of perfect control more.The composition more complicated of this Chinese medicine preparation, 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 its drug effect.Therefore the applicant formulated based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics finger-print and control the quality of injection based on the finger-print of fruit of Chinese magnoliavine composition characteristics comprehensively.But because contained complex chemical composition between each medicinal material in the pulse restoring injection causes interference to the formulation of finger-print, cause each several part finger-print feature instability, thus must control moving phase isochromatic spectrum condition, just can obtain good finger-print.That is to say, the finger-print of formulation of ' Sheng Mai ' is not that the finger-print simple superposition of red ginseng or genseng, the tuber of dwarf lilyturf and schisandra chinensis medicinal material or preparation is just getable, because three kinds of medicinal material ingredients disturbing effect each other in the prescription, cause the finger-print characteristic peak of red ginseng in the formulation of ' Sheng Mai ' or genseng and tuber of dwarf lilyturf part, fruit of Chinese magnoliavine part to change, and have only the condition of the present invention of employing, just can obtain desirable finger-print.
Proof by experiment, detection method of the present invention is more effective to the quality control of the Chinese medicine injection products made with red ginseng or genseng, the tuber of dwarf lilyturf and the fruit of Chinese magnoliavine, and method precision, stability are all higher.
Experimental example 1 based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics the preparation of finger-print
A, experimental apparatus, reagent and sample:
Reference substance: ginsenoside Rg
1: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
B, chromatographic condition and system flexibility 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 filling agent, tried out Zorbax, Inertsil ODS-3 respectively, Diamonsil ODS (is C18,4.6mm * 200mm, 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 DiamonsilODS chromatographic column (4.6mm * 200mm, 5 μ m) for use is the experimental study post.
2. the selection of moving phase:
Investigated (1) methanol-water (20: 80) in the research process respectively, (2) acetonitrile-water (10: 90), (3) acetonitrile-water (gradient elution) (4) A is 50mmol.L
-1KH
2PO
4Solution, B is that (the gradient elution volume proportion is from 0 minute to 5 minutes to acetonitrile-water (80: 20), 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%) four kinds of flow phase system.The result shows that peak shape is relatively poor under moving 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 moving phase (2) the moving 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 moving 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 moving 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 Agilent 1100 series of high efficiency liquid chromatographs 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 correlativity 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 red ginseng or genseng main active, its integral area proportion in finger-print more greatly and more stable is taken into account the research of intermediate and medicinal material simultaneously, therefore selected ginsenoside Rg
1As object of reference.
7. finger-print and technical parameter:
Formulate standard finger-print according to 10 batch samples, test sample finger-print and standard finger-print are compared, similarity is all between 0.90～1.00.
Experimental example 2 is based on the preparation of the finger-print of fruit of Chinese magnoliavine composition characteristics
A, experimental apparatus, reagent and sample:
Reference substance: schizandrin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
B, chromatographic condition and system flexibility 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 filling agent, 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 schizandrin).So finally selecting DiamonsilODS chromatographic column (250mm * 4.6mm, 5 μ m) for use is the experimental study post.
2. the selection of moving phase:
Investigated (1) methanol-water (20: 80) 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 methyl alcohol is 68%, from 6 minutes to 8 minutes, the ratio of methyl alcohol rises to 76% by 68%, from 8 minutes to 10 minutes, the ratio of methyl alcohol is 76%, from 10 minutes to 15 minutes, the ratio of methyl alcohol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methyl alcohol was 68%) four kinds of flow phase system.The result shows that peak shape is relatively poor under moving 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 moving phase (2) the moving 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 moving 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) moving 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 Agilent 1100 series of high efficiency liquid chromatographs 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 correlativity 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:
Schizandrin be in the fruit of Chinese magnoliavine main active it, its integral area proportion in finger-print more greatly and more stable is taken into account the research of intermediate and medicinal material simultaneously, therefore selected schizandrin is as object of reference.
7. finger-print and technical parameter:
Formulate standard finger-print according to 10 batch samples, test sample finger-print and standard finger-print are compared, similarity is all between 0.90～1.00.
The thin-layer chromatography discrimination method of the fruit of Chinese magnoliavine, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B in experimental example 3 pulse restoring injections:
Feature for the outstanding fruit of Chinese magnoliavine, selected the fruit of Chinese magnoliavine, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B is as its feature spot, but owing to exist more and schizandrin in the medicinal material, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B structure is close, the composition that polarity is similar, usual terms is difficult to reach requirements for quality control, so we have screened following thin layer plate and unfolding condition to fruit of Chinese magnoliavine schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B launches:
| Condition | Problem |
| Normal hexane-toluene-methanol-water (5: 5: 10: 4) silica gel H thin layer plate | Reference substance is expanded to the forward position |
| Cyclohexane-benzene-ethanol-formic acid (5: 5: 5: 3) silica gel H thin layer plate | Reference substance does not separate, and feminine gender has interference |
| With sherwood oil (30～60 ℃)-ethyl formate-methyl alcohol (10: 10: 3) silica gel g thin-layer plate | Reference substance is expanded to the forward position |
| With sherwood oil (60～90 ℃)-ethyl acetate-methyl alcohol (15: 5: 1) silica G F 254Thin layer plate | Reference substance does not separate, and feminine gender has interference |
| Toluene-methyl alcohol (10-5) silica gel H thin layer plate | Reference substance does not separate, and feminine gender has interference |
| Benzene-methyl alcohol (15-5) silica gel g thin-layer plate | Reference substance does not separate, and feminine gender has interference |
| Upper solution silica G F with sherwood oil (30～60 ℃)-ethyl formate-formic acid (15: 5: 1) 254Thin layer plate | It is clear to separate, and the moderate feminine gender of Rf value is noiseless |
Through screening, determined with the silica gel g thin-layer plate to be stationary phase, upper solution with sherwood oil (30～60 ℃)-ethyl formate-formic acid (15: 5: 1) is a developping agent, with this understanding, the Rf value of schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B is moderate, it is clear to separate with other spot, negative noiseless.
The liquid chromatography discrimination method of schizandrin, schizandrin A, deoxyschizandrin in experimental example 4 pulse restoring injections:
Feature for the outstanding fruit of Chinese magnoliavine, except the thin layer discrimination method, selected schizandrin, schizandrin A, deoxyschizandrin as its characteristic component, but owing to have more and schizandrin, schizandrin A, the deoxyschizandrin structure is close, polarity is similar composition in the medicinal material, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with moving phase schizandrin, schizandrin A, deoxyschizandrin are separated:
Through screening, determined with the octadecylsilane chemically bonded silica to be stationary phase, methanol-water (65: 35) be a moving phase, and with this understanding, schizandrin, schizandrin A, deoxyschizandrin retention time are moderate, and the peak is capable sharp-pointed, symmetry, feminine gender is noiseless.
The thin-layer chromatography discrimination method of the tuber of dwarf lilyturf in experimental example 5 pulse restoring injections:
Feature for the outstanding tuber of dwarf lilyturf, selected tuber of dwarf lilyturf control medicinal material as its feature spot, but owing to have the composition that more structure is close, polarity is similar in the medicinal material, usual terms is difficult to reach requirements for quality control, so we have screened following thin layer plate and unfolding condition to launching the tuber of dwarf lilyturf:
| Condition | Problem |
| Benzene-methyl alcohol-ethyl acetate (50: 10: 3) silica G F 254Thin layer plate | Reference substance does not separate, and feminine gender has interference |
| Toluene-methyl alcohol-methyl acetate (50: 10: 3) silica gel H thin layer plate | Reference substance does not separate, and feminine gender has interference |
| Chloroform-normal hexane-formic acid (50: 20: 5) silica gel g thin-layer plate | Reference substance is expanded to the forward position |
| Methylene chloride-cyclohexane (5: 2) silica gel g thin-layer plate | Reference substance is expanded to the forward position |
| Ethyl acetate-pyridine-acetone (5: 3: 5) silica gel H thin layer plate | Reference substance does not separate, and feminine gender has interference |
| Benzene-ethyl formate-formic acid (60: 10: 2) silica gel g thin-layer plate | Reference substance does not separate, and feminine gender has interference |
| Toluene-methyl alcohol-glacial acetic acid (80: 5: 0.1) silica G F 254Thin layer plate | It is clear to separate, and the moderate feminine gender of Rf value is noiseless |
Through screening, determined with silica G F
254Thin layer plate is a stationary phase, is developping agent with toluene-methyl alcohol-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 spot, and is negative noiseless.
Ophiopogonin B in experimental example 6 pulse restoring injections, 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 spot, but owing to have more and ophiopogonin B, ophiopogonin D, ophiopogonin D ' structure is close, polarity is similar composition in the medicinal material, usual terms is difficult to reach requirements for quality control, so we have screened following thin layer plate and unfolding condition to launching the tuber of dwarf lilyturf:
| Condition | Problem |
| Ethyl acetate-methyl alcohol-formic acid (10: 10: 0.5) silica gel g thin-layer plate | Reference substance is expanded to the forward position |
| Methenyl choloride-ethanol-glacial acetic acid (10: 10: 0.5) silica gel H thin layer plate | Reference substance is expanded to the forward position |
| Ethyl acetate-methyl alcohol-acetone (10: 3: 0.2) silica gel g thin-layer plate | Reference substance does not separate, and feminine gender has interference |
| Methylene chloride-methanol-normal hexane (15: 10: 2) silica G F 254Thin layer plate | Reference substance is expanded to the forward position |
| Ethyl acetate-methyl alcohol-butanone (20: 3: 1) silica G F 254Thin layer plate | Reference substance does not separate, and feminine gender has interference |
| Ethyl acetate-methyl alcohol-cyclohexane (20: 8: 1) silica gel g thin-layer plate | Reference substance does not separate, and feminine gender has interference |
| With ethyl acetate-methanol-water (15: 5: 1) silica gel g thin-layer plate | It is clear to separate, and the moderate feminine gender of Rf value is noiseless |
Through screening, determined with the silica gel g thin-layer plate to be stationary phase, be developping agent with ethyl acetate-methanol-water (15: 5: 1), with this understanding, the Rf value of ophiopogonin B, ophiopogonin D, ophiopogonin D ' spot is moderate, and it is clear to separate with other spot, and is negative noiseless.
Ophiopogonin B in experimental example 7 pulse restoring injections, ophiopogonin D, ophiopogonin D ' the liquid chromatography discrimination method:
Feature for outstanding ophiopogonin, except the thin layer discrimination method, ophiopogonin B, ophiopogonin D, ophiopogonin D ' have been selected as its characteristic component, but owing to have more and ophiopogonin B, ophiopogonin D, ophiopogonin D ' structure is close, polarity is similar composition in the medicinal material, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and moving phase to ophiopogonin B, ophiopogonin D, ophiopogonin D ' separate:
| Condition | Problem |
| Methyl alcohol-0.05mol/L sodium dihydrogen phosphate (99: 1) octadecylsilane chemically bonded silica | Appearance time is too fast |
| Acetonitrile-0.05mol/L sodium dihydrogen phosphate (95: 5) octadecylsilane chemically bonded silica | Appearance time is too fast |
| Methyl alcohol-0.02mol/L sodium hydrogen phosphate (95: 5) eight alkyl silane bonded silica gels | Feminine gender has interference |
| Methyl alcohol-0.02mol/L potassium dihydrogen phosphate (95: 5) octadecylsilane chemically bonded silica | Feminine gender has interference |
| Acetonitrile-0.02mol/L sodium hydrogen phosphate (95: 5) eight alkyl silane bonded silica gels | There is bifurcated at the peak, |
| Acetonitrile-tetrahydrofuran-1% glacial acetic acid (80: 10: 10) octadecylsilane chemically bonded silica | Feminine gender has interference |
| Acetonitrile-water (90: 10) octadecylsilane chemically bonded silica | Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless |
Through screening, determined with the octadecylsilane chemically bonded silica to be stationary phase, acetonitrile-water (90: 10) be a moving 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 experimental example 8 pulse restoring injections:
Feature for outstanding ophiopogonin unit, selected Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin as its feature spot, but owing to have more and Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin structure is close, polarity is similar composition in the medicinal material, usual terms is difficult to reach requirements for quality control, so we have screened following thin layer plate and unfolding condition to launching the tuber of dwarf lilyturf:
| Condition | Problem |
| Methyl alcohol-chloroform-glacial acetic acid (5: 3: 2) silica gel g thin-layer plate | Reference substance is expanded to the forward position |
| Normal hexane-ethyl acetate-glacial acetic acid (5: 3: 1) silica gel H thin layer plate | Reference substance is expanded to the forward position |
| Normal hexane-ethyl formate (3: 2) silica G F 254Thin layer plate | Feminine gender has interference |
| Methyl alcohol-ethyl acetate-formic acid (2: 1: 1) silica gel g thin-layer plate | Feminine gender has interference |
| Methyl alcohol-ethyl acetate-acetone (1: 1: 1) silica gel H thin layer plate | Feminine gender has interference |
| Methyl alcohol-ethyl formate (2: 1.5) silica gel H thin layer plate | Feminine gender has interference |
| Normal hexane-ethyl acetate-water (1: 1: 1) silica gel g thin-layer plate | It is clear to separate, and the moderate feminine gender of Rf value is noiseless |
Through screening, determined with the silica gel g thin-layer plate to be stationary phase, be developping agent 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 spot, and is negative noiseless.
The liquid chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in experimental example 9 pulse restoring injections
Feature for outstanding ophiopogonin unit, except the thin layer discrimination method, selected Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin as its characteristic component, but owing to have more and Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin structure is close, polarity is similar composition in the medicinal material, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with moving phase Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin are separated:
Through screening, determined with the octadecylsilane chemically bonded silica to be stationary phase, acetonitrile-water (90: 10) is a moving 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 experimental example 10 pulse restoring injections
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin-layer chromatography discrimination method
For the feature of outstanding red ginseng or genseng, selected the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf be as its feature spot, but owing to exist more and the ginsenoside Rg in the medicinal material
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's structure is close, polarity is similar composition, usual terms is difficult to reach requirements for quality control, so we have screened following thin layer plate and unfolding condition to launching the tuber of dwarf lilyturf:
| Condition | Problem |
| Lower floor's solution silica gel g thin-layer plate that chloroform-normal hexane-water (20: 60: 10) is placed below 10 ℃ | Reference substance is expanded to the forward position |
| Methylene chloride-ethyl formate-cyclohexane (10: 1: 5) silica gel H thin layer plate | Reference substance is expanded to the forward position |
| Chloroform-butanone-methyl alcohol (15: 40: 22) silica gel g thin-layer plate | Reference substance does not separate, and feminine gender has interference |
| Lower floor's solution silica gel g thin-layer plate that chloroform-ethyl acetate-water (10: 40: 15) is placed below 10 ℃ | Reference substance does not separate, and feminine gender has interference |
| Normal butyl alcohol-ethyl formate-water (5: 1: 5) upper solution silica gel g thin-layer plate | Reference substance does not separate, and feminine gender has interference |
| Isopropyl alcohol-ethyl formate-water (2: 1.5: 0.5) upper solution silica gel H thin layer plate | Reference substance does not separate, and feminine gender has interference |
| Chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower floor's solution silica gel g thin-layer plate of placing below 10 ℃ | It is clear to separate, and the moderate feminine gender of Rf value is noiseless |
Through screening, determined with the silica gel g thin-layer plate to be stationary phase, with chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developping agent, with this understanding, the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the Rf value moderate, it is clear to separate with other spot, negative noiseless.
Ginsenoside Rg in experimental example 11 pulse restoring injections
1, ginsenoside Rb
1, the ginsenoside Re the liquid chromatography discrimination method
For the feature of outstanding red ginseng or genseng, except the thin layer discrimination method, 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 medicinal material
1, ginsenoside Rb
1, ginsenoside Re's structure is close, polarity is similar composition, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and moving phase to the ginsenoside Rg
1, ginsenoside Rb
1, the ginsenoside Re separates:
| Condition | Problem |
| Methyl alcohol-tetrahydrofuran-water (85: 5: 10) octadecylsilane chemically bonded silica | Appearance time is too fast |
| Acetonitrile-tetrahydrofuran water (90: 5: 5) octadecylsilane chemically bonded silica | Appearance time is too fast |
| Methyl alcohol-0.005mol/L sodium dihydrogen phosphate (80: 20) eight alkyl silane bonded silica gels | Feminine gender has interference |
| Methyl alcohol-0.02mol/L sodium hydrogen phosphate (80: 20) octadecylsilane chemically bonded silica | Feminine gender has interference |
| Acetonitrile-0.005mol/L sodium dihydrogen phosphate ((90: 10) eight alkyl silane bonded silica gels | Feminine gender has interference |
| Acetonitrile-0.05mol/L potassium dihydrogen phosphate ((85: 15) octadecylsilane chemically bonded silica | Feminine gender has interference |
| Acetonitrile-water is a moving 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% octadecylsilane chemically bonded silica by 29% | Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless |
Through screening, determined with the octadecylsilane chemically bonded silica to be stationary phase, acetonitrile-water is a moving 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.
Ginsenoside Rg in experimental example 12 pulse restoring injections
1, ginsenoside Rb
1, the ginsenoside Re content assaying method
1. instrument and reagent (1) instrument: Agilent1100 high performance liquid chromatograph, chemstationsys workstation.The TU-1800SPC ultraviolet spectrophotometer.
(2) reagent: ginsenoside Rg
1, ginsenoside Rb
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, ginsenoside Rb
1, the ginsenoside Re, split in the 10ml measuring bottle, add methyl alcohol dilution and make the solution that every 1ml contains 0.5mg, scan in the 200-400nm wavelength coverage.The ginsenoside Rg
1, ginsenoside Rb
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); Moving phase: acetonitrile-water is a moving 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 Rg
1, ginsenoside Rb
1An amount of with the ginsenoside Re, accurate claim surely, add methyl alcohol and make 1ml and contain the ginsenoside Rg
10.30mg, ginsenoside Rb
10.30mg, the mixed solution of ginsenoside Re 0.2mg.
5. the preparation of need testing solution: get under the content uniformity item 5 bottles of this product, get content, mixing is therefrom got about 0.5g, accurately claims surely, puts in the 5ml measuring bottle, adds and flows mutual-assistance dissolving and be diluted to scale, shakes up, promptly.
With this understanding, negative sample ginsenoside Rg in the disturbed specimen not
1, ginsenoside Rb
1With ginsenoside Re's mensuration, and degree of separation is good.
6. ginsenoside Rg
1, ginsenoside Rb
1Take by weighing the ginsenoside Rg with ginsenoside Re's linear relationship precision
110.24mg, ginsenoside Rb
110.09mg, ginsenoside Re 7.54mg, put altogether in the 10ml measuring bottle, it is fixed to scale to add moving phase, shakes up, product solution (contains the ginsenoside Rg among every 1ml in contrast
11.024mg, ginsenoside Rb
11.009mg, contain ginsenoside Re 0.754mg), precision is measured 2.5ml, puts in the 10ml measuring bottle, adds moving phase to scale, shakes up, in contrast the product dilute solution.(contain the ginsenoside Rg among every 1ml
10.256mg, contain ginsenoside Rb
10.25225mg, contain ginsenoside Re 0.1885mg) 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 liquid chromatograph, is ordinate with the peak area, and ginsenoside Rg1 and ginsenoside Re's amount is figure for horizontal ordinate, the drawing standard curve.
The ginsenoside Rg
1Linear relationship
| Numbering | The ginsenoside Rg 1(μg) | Peak area |
| 1 | 0.512 | 150.63 |
| 2 | 1.280 | 475.54 |
| 3 | 2.560 | 890.23 |
| 4 | 3.072 | 1058.85 |
| 5 | 6.144 | 2107.02 |
| 6 | 10.24 | 3538.96 |
Regression equation: Y=345.01X+1.20;
The coefficient of determination: γ=0.9996;
The ginsenoside Rg
1Good in 0.512～10.240 μ g scope internal linear relation.
Ginsenoside Rb
1Linear relationship
| Numbering | Ginsenoside Rb 1(μg) | Peak area |
| 1 | 0.5045 | 142.08 |
| 2 | 1.2613 | 353.07 |
| 3 | 2.5225 | 715.48 |
| 4 | 3.0270 | 860.21 |
| 5 | 6.0540 | 1709.14 |
| 6 | 10.090 | 2855.26 |
Regression equation: Y=282.95X-0.44;
The coefficient of determination: γ=0.9999;
Ginsenoside Rb
1Good in 0.5045～10.090 μ g scope internal linear relation.
Ginsenoside Re's linear relationship
| Numbering | Ginsenoside Re (μ g) | Peak area |
| 1 | 0.3770 | 184.37 |
| 2 | 0.9425 | 466.91 |
| 3 | 1.8850 | 928.26 |
| 4 | 2.2620 | 1104.76 |
| 5 | 4.5240 | 2216.15 |
| 6 | 7.5400 | 3698.24 |
Regression equation: Y=490.12X+1.11;
The coefficient of determination: γ=0.9999;
The ginsenoside Re is good in 0.3770～7.5400 μ g scope internal linear relation.
7. reference substance precision and stability test precision is drawn ginsenoside Rg under the typical curve item
1, the ginsenoside Rg
1(contain the ginsenoside Rg among every 1ml with ginsenoside Re's reference substance dilute solution
10.256mg, ginseng saponin(e Rb
10.25225mg, contain ginsenoside Re 0.1885mg) and 10 μ l, inject liquid chromatograph, the record peak area is measured at 0,6,12,24,48 hour sample introduction.
The test of reference substance solution precision
The result shows, the ginsenoside Rg
1, ginsenoside Rb
1With ginsenoside Re's reference substance solution precision and having good stability.
5 bottles of this product are got under the weight differential item in 8 need testing solution stability tests, get content, mixing is therefrom got about 0.5g, and accurate the title decides, put in the 5ml measuring bottle, add and flow mutual-assistance dissolving and decide to shake up the accurate 10 μ l that draw to scale, inject liquid chromatograph, measure at 0,6,12,24,48 hour sample introduction respectively.
Need testing solution stability test result
| Test duration (h) | 0 | 6 | 12 | 24 | 48 | Average | RSD(％) |
| The ginsenoside Rg 1(mg/ bottle) | 1.06 | 1.02 | 1.04 | 1.03 | 1.05 | 1.04 | 1.52 |
| Ginsenoside Rb 1(mg/ bottle) | 0.99 | 1.00 | 0.97 | 1.01 | 0.98 | 0.99 | 1.60 |
| Ginsenoside Re's (mg/ bottle) | 0.52 | 0.54 | 0.53 | 0.53 | 0.54 | 0.53 | 1.57 |
The result shows that need testing solution has good stability.
9. replica test is got under this product content uniformity item 5 bottles on powder pin, get content, porphyrize is therefrom got about 0.5g (totally 5 parts), the accurate title, decide, split in the 5ml measuring bottle, add the mutual-assistance dissolving and fixed of flowing, shake up to scale, the accurate 10 μ l that draw, inject liquid chromatograph, calculate with one point external standard method, promptly.
Replica test
| Test number | 1 | 2 | 3 | 4 | 5 | Average | RSD(％) |
| The ginsenoside Rg 1(mg/ bottle) | 1.08 | 1.11 | 1.07 | 1.10 | 1.09 | 1.09 | 1.45 |
| Ginsenoside Rb 1(mg/ bottle) | 1.02 | 1.04 | 1.01 | 1.03 | 1.02 | 1.02 | 1.11 |
| Ginsenoside Re's (mg/ bottle) | 0.55 | 0.54 | 0.54 | 0.55 | 0.53 | 0.54 | 1.54 |
10. the application of sample absorption method is adopted in the average recovery test, gets this product under the weight differential item, gets content, and mixing is therefrom got about 250mg (totally 5 parts), and accurate the title decides, and splits in the 5ml measuring bottle; Precision takes by weighing the ginsenoside Rg
111.58mg, ginsenoside Rb
110.84mg ginsenoside Re 5.76mg puts in the 25ml measuring bottle altogether, add an amount of sonicated of moving phase and make dissolving, take out, put to room temperature, add moving phase to scale, shake up, precision is measured 1ml (totally 5 parts), split in the above-mentioned 5ml measuring bottle, add moving phase, shake up to scale, the accurate 10 μ l that draw, inject liquid chromatograph, calculate with one point external standard method, promptly.
Ginsenoside Rg in the pulse restoring injection
1Content: 0.1812%;
Ginsenoside Rb in the pulse restoring injection
1Content: 0.1695%;
Ginsenoside Re's content in the pulse restoring injection: 0.0897%;
The ginsenoside Rg
1The average recovery test
| Numbering | Test sample weighing (mg) | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 246.93 | 0.4474 | 0.4632 | 0.8974 | 97.15 |
| 2 | 255.12 | 0.4623 | 0.4632 | 0.9173 | 98.23 |
| 3 | 253.08 | 0.4586 | 0.4632 | 0.9055 | 96.49 |
| 4 | 249.47 | 0.4520 | 0.4632 | 0.9097 | 98.81 |
| 5 | 261.25 | 0.4734 | 0.4632 | 0.9270 | 97.94 |
The ginsenoside Rg
1Average recovery rate=97.72%; RSD=0.93%
Ginsenoside Rb
1The average recovery test
| Numbering | Test sample weighing (mg) | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 246.93 | 0.4185 | 0.4336 | 0.8446 | 98.25 |
| 2 | 255.12 | 0.4324 | 0.4336 | 0.8630 | 99.31 |
| 3 | 253.08 | 0.4290 | 0.4336 | 0.8541 | 98.04 |
| 4 | 249.47 | 0.4229 | 0.4336 | 0.8503 | 98.57 |
| 5 | 261.25 | 0.4428 | 0.4336 | 0.8601 | 96.23 |
Ginsenoside Rb
1Average recovery rate=98.08%; RSD=1.16%
The test of ginsenoside Re's average recovery
Ginsenoside Re's average recovery rate=96.93%; RSD=1.15%
11. three batches of pilot scale sample sizes are measured
Get injection and give birth to three batches in arteries and veins sample, press the described method of text and handle.
Three batch sample assay results
| Lot number | The ginsenoside Rg 1(mg/ bottle) | Ginsenoside Rb 1(mg/ bottle) | Ginsenoside Re's (mg/ bottle) |
| 1 | 1.12 | 0.97 | 0.55 |
| 2 | 1.08 | 1.03 | 0.52 |
| 3 | 1.14 | 1.06 | 0.58 |
From above test as can be known, adopt the method for the invention to measure ginsenoside Rg in the pulse restoring injection
1, ginsenoside Rb
1, the ginsenoside Re content precision, stability and repeatability good, recovery height, method is easy, data accurately, reliable.
General ginsenoside content assaying method in experimental example 13 pulse restoring injections
Instrument, reagent (1) instrument: the general logical TU-1810SPC ultraviolet/visible spectrophotometer of analysing
SARTORIUS BP211D electronic analytical balance
(2) reagent: vanillic aldehyde is analyzed pure Tianjin and is recovered fine chemistry industry research institute
Methyl alcohol chromatographically pure J.T.Baker
Glacial acetic acid is analyzed pure Shanghai reagent one factory
Perchloric acid is analyzed chemical plant, prosperous source, pure Tianjin
The 2 selection precisions that detect wavelength take by weighing the ginsenoside Rg
16.11mg, put in the 100ml measuring bottle, add an amount of methyl alcohol sonicated (power 250W, frequency 33KHz) makes dissolving, add methyl alcohol 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% vanillic aldehyde-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 scans in 700～400nm wavelength coverage, the result shows, the ginsenoside Rg
1At the 547nm place absorption maximum is arranged, blank noiseless, therefore selecting 547nm is the detection wavelength of general ginsenoside in the spectrophotometry pulse restoring injection.
The investigation precision of 3 linear relationships takes by weighing the ginsenoside Rg
1Reference substance 6.85mg puts in the 100ml measuring bottle, adds an amount of sonicated of methyl alcohol (power 250W, frequency 33KHZ) makes dissolving, take out, put to room temperature, add methyl alcohol 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% vanillic aldehyde-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 spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2000), measure absorbance log at the wavelength place of 547nm.With the absorbance log is ordinate, the ginsenoside Rg
1Amount (μ g) be horizontal ordinate, the drawing standard curve.
Regression equation Y=0.0049X-0.0077; R=0.999
The ginsenoside Rg
1Linear good in 27.4～137.0 μ g scopes.
The ginsenoside Rg
1The standard curve determination data
| Numbering | The ginsenoside Rg 1(μg) | Absorbance log |
| 1 | 27.4 | 0.121 |
| 2 | 54.8 | 0.261 |
| 3 | 82.2 | 0.404 |
| 4 | 109.6 | 0.534 |
| 5 | 137.0 | 0.656 |
4 precision test precision is measured the ginsenoside Rg
1Reference substance solution (0.0685mg/ml) 1.2ml puts in the 10ml tool plug test tube water bath method solvent, take out accurate 5% vanillic aldehyde-glacial acetic acid solution 0.2ml, the perchloric acid 0.8ml of adding, 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, shaking up, is blank with the retinue solvent, measures absorbance log according to spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2000) at the wavelength place of 547nm.With the absorbance log is ordinate, the ginsenoside Rg
1Amount (μ g) be horizontal ordinate, the drawing standard curve.
Regression equation Y=0.0049X-0.0077; R=0.999
The ginsenoside Rg
1Linear good in 27.4～137.0 μ g scopes.
The ginsenoside Rg
1The standard curve determination data
| Numbering | The ginsenoside Rg 1(μg) | Absorbance log |
| 1 | 27.4 | 0.121 |
| 2 | 54.8 | 0.261 |
| 3 | 82.2 | 0.404 |
| 4 | 109.6 | 0.534 |
| 5 | 137.0 | 0.656 |
4 precision test precision is measured the ginsenoside Rg
1Reference substance solution (0.0685mg/ml) 1.2ml puts in the 10ml tool plug test tube water bath method solvent, take out accurate 5% vanillic aldehyde-glacial acetic acid solution 0.2ml, the perchloric acid 0.8ml of adding, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, cooled off 2 minutes in ice-water bath immediately, the accurate glacial acetic acid 5ml that adds shakes up, with the retinue solvent is blank, according to spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2000), measure absorbance log, METHOD FOR CONTINUOUS DETERMINATION 5 times at the wavelength place of 547nm.
The precision experiment
| 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 |
5 stability tests
5.1 the stability test precision of reference substance solution is measured the ginsenoside Rg
1Reference substance solution (0.0727mg/ml) 1.2ml, put in the 10ml tool plug test tube, the water bath method solvent, take out, the accurate 5% vanillic aldehyde-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml shakes up, and puts in 60 ℃ of water-baths and heats 15 minutes, take out, cooled off 2 minutes in ice-water bath immediately, the accurate glacial acetic acid 5ml that adds shakes up, with the retinue solvent is blank, according to spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2000), measure absorbance log at the wavelength place of 547nm, in the time of 0,10,20,40,60 minute, measure respectively.
The reference substance solution stability experiment
| Time (min) | 0 | 10 | 20 | 40 | 60 | Average | RSD％ |
| Absorbance | 0.453 | 0.453 | 0.450 | 0.451 | 0.449 | 0.451 | 0.40 |
5.2 the stability experiment of need testing solution is got under this product content uniformity item 5 bottles on powder pin, gets content, porphyrize, therefrom get about 100mg, the accurate title, decide, and puts in the 10ml measuring bottle, adds water and make dissolving and fixed to scale in right amount, shake up, precision is measured 0.2ml, puts in the 10ml tool plug test tube water bath method solvent, take out, the accurate 5% vanillic aldehyde-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, with the retinue solvent is blank, according to spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2000), measures absorbance log at the wavelength place of 547nm, calculate with one point external standard method, respectively 0,10,20,40, measure in the time of 60 minutes.
The need testing solution stability experiment
| Time (min) | 0 | 10 | 20 | 40 | 60 | Average | RSD％ |
| Content (mg/ bottle) | 22.18 | 21.74 | 22.03 | 21.59 | 22.55 | 22.02 | 1.71 |
6 replica tests are got under this product content uniformity item 5 bottles on powder pin, get content, porphyrize is therefrom got about 100mg (totally 5 parts), split in the 10ml measuring bottle, add water and make dissolving and fixed to scale in right amount, shake up, precision is measured 0.2ml, split in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillic aldehyde-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 spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2000), wavelength place at 547nm measures absorbance log, calculates with one point external standard method, promptly.
Repeated experiment
7 recovery tests adopt the application of sample absorption method, get under this product content uniformity item 5 bottles on powder pin, get content, and porphyrize is therefrom got about 50mg (totally 5 parts), and accurate the title splits in the 10ml measuring bottle calmly; Other gets the ginsenoside Rg
1The about 1.8mg of reference substance (totally 5 parts), the accurate title, decide, split in the above-mentioned 10ml measuring bottle, add water and make dissolving and fixed in right amount, shake up to scale, precision is measured 0.2ml, putting in the 10ml tool plug test tube, by operating under the text determination method item, is blank with the retinue solvent, measure absorbance log in accordance with the law, read ginsenoside Rg the need testing solution from typical curve
1Content, calculate with one point external standard method, promptly.
The content of total saponins in the pulse restoring injection: 3.666%
The experiment of general ginsenoside average recovery
| Numbering | Test sample weighing (mg) | Total saponins amount (mg) in the test sample | The ginsenoside Rg 1Addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 50.24 | 1.842 | 1.77 | 3.581 | 98.25 |
| 2 | 47.81 | 1.753 | 1.92 | 3.628 | 97.66 |
| 3 | 55.23 | 2.025 | 2.13 | 4.140 | 99.32 |
| 4 | 50.94 | 1.867 | 2.04 | 3.876 | 98.47 |
| 5 | 51.67 | 1.894 | 1.86 | 3.697 | 96.93 |
Average recovery rate=99.53% RSD=1.88%
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.
Three batch sample assay results
| Lot number | General ginsenoside (mg/ bottle) |
| 1 | 22.17 |
| 2 | 22.39 |
| 3 | 21.94 |
From above test as can be known, it is good to adopt the method for the invention to measure the content precision of general ginsenoside in the pulse restoring injection, stability and repeatability, recovery height, and method is easy, and data are accurate, reliable.
The content assaying method of schizandrin, schizandrin A, deoxyschizandrin in experimental example 14 pulse restoring injections
1. instrument and reagent
(1) instrument: Agilent1100 high performance liquid chromatograph, chemstationsys workstation; The TU-1800SPC ultraviolet spectrophotometer; The AE240 electronic balance.
(2) reagent: schizandrin, schizandrin A, deoxyschizandrin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
2. it is an amount of that the selection precision that detects wavelength takes by weighing schizandrin, schizandrin A, deoxyschizandrin reference substance, add methyl alcohol respectively and be diluted to scale, shake up, get every 1ml and contain schizandrin 0.10mg respectively, schizandrin A 0.10mg, the solution of deoxyschizandrin 0.10mg scans in the 200-400nm wavelength coverage respectively.The result shows: schizandrin, schizandrin A, deoxyschizandrin all have absorption maximum at the 250nm place, therefore select the detection wavelength of 250nm as assay.
3. chromatographic condition: Dikma ODS (4.6 * 250mm, 5um); Moving phase: methanol-water (65: 35) is a moving phase; The detection wavelength is 250nm; Flow velocity: 1.0ml/min.
4. system suitability test
4.1 it is an amount of that the preparation precision of reference substance solution takes by weighing schizandrin, schizandrin A, deoxyschizandrin reference substance, adding methyl alcohol makes every 1ml and contains schizandrin 0.02mg, schizandrin A 0.003mg, the reference substance mixed solution of deoxyschizandrin 0.005mg, promptly.
4.2 the preparation of need testing solution is got shengmai injection as need testing solution.
4.3 the preparation of negative need testing solution takes by weighing the negative sample that does not contain the fruit of Chinese magnoliavine, according to preparation method's 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 liquid chromatograph, measure the record chromatogram.As seen from the figure, schizandrin in the test sample, deoxyschizandrin, schizandrin A peak separate good, and it is clear to separate with close peak, and fully, schizandrin, schizandrin A in the negative test sample, the deoxyschizandrin peak is noiseless.
5. linear relationship is investigated precision and is taken by weighing schizandrin reference substance 10.27mg, schizandrin A reference substance 10.03mg, deoxyschizandrin reference substance 9.86mg, split in the 50ml measuring bottle, add methyl alcohol respectively and make dissolving and fixed to scale in right amount, shake up, precision is measured schizandrin reference substance solution 2ml, schizandrin A reference substance solution 0.3ml, deoxyschizandrin reference substance solution 0.6ml puts in the 10ml measuring bottle altogether, and it is fixed to scale to add methyl alcohol, shake up, the accurate 2 μ l that draw, 4 μ l, 6 μ l, 8 μ l, 10 μ l inject liquid chromatograph, are ordinate with the peak area, sample size (μ g) is figure for horizontal ordinate, the drawing standard curve.
The schizandrin linear relationship is investigated
| Numbering | Schizandrin (μ g) | Peak area |
| 1 | 0.08216 | 121.45 |
| 2 | 0.16432 | 238.71 |
| 3 | 0.24648 | 362.58 |
| 4 | 0.32864 | 487.62 |
| 5 | 0.41080 | 601.91 |
Regression equation: Y=1472.5X-0.495;
The coefficient of determination: γ=0.9998;
Schizandrin is good in 0.08216～0.4108 μ g scope internal linear relation.
The schizandrin A linear relationship is investigated
| Numbering | Schizandrin A (μ g) | Peak area |
| 1 | 0.012036 | 121.36 |
| 2 | 0.024072 | 245.71 |
| 3 | 0.036108 | 371.96 |
| 4 | 0.048144 | 494.18 |
| 5 | 0.060180 | 610.42 |
Regression equation: Y=10191X+0.749;
The coefficient of determination: γ=0.9999;
Schizandrin A is good in 0.012036～0.060180 μ g scope internal linear relation.
The deoxyschizandrin linear relationship is investigated
| Numbering | Deoxyschizandrin (μ g) | Peak area |
| 1 | 0.023664 | 184.61 |
| 2 | 0.047328 | 381.09 |
| 3 | 0.070992 | 561.82 |
| 4 | 0.094656 | 745.96 |
| 5 | 0.118320 | 940.83 |
Regression equation: Y=7993.2X-0.331;
The coefficient of determination: γ=0.9999;
Deoxyschizandrin is good in 0.023664～0.11832 μ g scope internal linear relation.
6. precision test precision is drawn reference substance mixed solution (every 1ml contains schizandrin 20.54 μ g respectively, schizandrin A 3.009 μ g, deoxyschizandrin 5.916 μ g) 10 μ l, inject liquid chromatograph, the record peak area, METHOD FOR CONTINUOUS DETERMINATION 5 times, the precision of investigation reference substance solution.
The test of reference substance precision
The result shows that reference substance solution precision is good.
7. the accurate shengmai injection 10 μ l that draw of stability test inject liquid chromatograph, at 0,6,12,24,48 hour sample introduction, calculate with one point external standard method, promptly respectively.
The reference substance solution stability test
| Time (h) | 0 | 6 | 12 | 24 | 48 | Average | RSD(％ ) |
| Schizandrin (mg/ props up) | 0.2514 | 0.2547 | 0.2533 | 0.2529 | 0.2568 | 0.2538 | 0.80 |
| Schizandrin A (mg/ props up) | 0.0338 | 0.0331 | 0.0336 | 0.0341 | 0.0339 | 0.0337 | 1.13 |
| Deoxyschizandrin (mg/ props up) | 0.0658 | 0.0642 | 0.0666 | 0.0675 | 0.0651 | 0.0658 | 1.95 |
The result shows that reference substance solution has good stability.
8. the accurate shengmai injection 10 μ l that draw of replica test inject liquid chromatograph, calculate with one point external standard method, promptly.
Replica test
| Numbering | 1 | 2 | 3 | 4 | 5 | Average | RSD (％ ) |
| Schizandrin (mg/ props up) | 0.2528 | 0.2503 | 0.2519 | 0.2542 | 0.2581 | 0.2535 | 1.17 |
| Schizandrin A (mg/ props up) | 0.0342 | 0.0335 | 0.0351 | 0.0346 | 0.0347 | 0.0344 | 1.76 |
| Deoxyschizandrin (mg/ props up) | 0.0662 | 0.0657 | 0.0682 | 0.0673 | 0.0665 | 0.0668 | 1.47 |
The result shows that repeatability is good.
9. the application of sample absorption method is adopted in the average recovery test, and precision is measured shengmai injection 5ml, splits in the 10ml measuring bottle; Precision is measured reference substance mixed solution (every 1ml contains schizandrin 0.1044mg respectively, schizandrin A 0.01446mg, deoxyschizandrin 0.02552mg) 1ml (totally 5 parts), split in the above-mentioned 10ml measuring bottle, add the about 6ml of methyl alcohol, jolting adds methyl alcohol to scale, shake up, filter, the accurate subsequent filtrate 10 μ l that draw inject liquid chromatograph, calculate with one point external standard method, promptly.
Schizandrin content in the shengmai injection: 0.2535mg/ props up;
Schizandrin A content in the shengmai injection: 0.0344mg/ props up;
Deoxyschizandrin content in the shengmai injection: 0.0668mg/ props up.
The test of schizandrin average recovery
| Numbering | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 0.12675 | 0.1044 | 0.2293 | 98.25 |
| 2 | 0.12675 | 0.1044 | 0.2301 | 99.03 |
| 3 | 0.12675 | 0.1044 | 0.2306 | 99.47 |
| 4 | 0.12675 | 0.1044 | 0.2282 | 97.19 |
| 5 | 0.12675 | 0.1044 | 0.2307 | 99.56 |
Schizandrin average recovery rate=98.70%; RSD=1.00%.
The test of schizandrin A average recovery
| Numbering | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 0.0172 | 0.01446 | 0.03115 | 96.48 |
| 2 | 0.0172 | 0.01446 | 0.03125 | 97.15 |
| 3 | 0.0172 | 0.01446 | 0.03152 | 99.01 |
| 4 | 0.0172 | 0.01446 | 0.03128 | 97.34 |
| 5 | 0.0172 | 0.01446 | 0.03141 | 98.25 |
Schizandrin A average recovery rate=97.65%; RSD=1.01%.
The test of deoxyschizandrin average recovery
Deoxyschizandrin average recovery rate=96.62%; RSD=1.19%.
10. three batches of pilot scale sample sizes are measured
Get three batches in this product sample, press the described method of text and handle.
Three batch sample assay results
| Lot number | 1 | 2 | 3 |
| Schizandrin (mg/ props up) | 0.2541 | 0.2551 | 0.2536 |
| Schizandrin A (mg/ props up) | 0.0351 | 0.0344 | 0.0352 |
| Deoxyschizandrin (mg/ props up) | 0.0664 | 0.0672 | 0.0668 |
From above test as can be known, it is good to adopt the method for the invention to measure the content precision of schizandrin, schizandrin A, deoxyschizandrin in the shengmai injection, stability and repeatability, recovery height, and method is easy, and data are accurate, reliable.
The assay of total lignan in experimental example 15 pulse restoring injections
1 instrument and reagent
(1) key instrument:
Ultraviolet spectrophotometer TU-1810SPC Beijing Puxi General Instrument Co., Ltd
Electronic analytical balance BP211D SARTORIUS
Supersonic wave cleaning machine KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.
(2) reagent:
Schizandrin Nat'l Pharmaceutical ﹠ Biological Products Control Institute
Ethanol is analyzed pure atropic Fine Chemical Co., Ltd now
The schizandrin reference substance is got in 2 selections that detect wavelength, adds methyl alcohol and makes the solution that every 1ml contains 20 μ g, in contrast product solution.Measure shengmai injection 4ml, put in the 50ml measuring bottle, add an amount of sonicated of methanol solution (power 250W, frequency 33KHz) and make dissolving, take out, put, be diluted to scale, shake up, filter, get subsequent filtrate as need testing solution with methanol solution to room temperature.Draw the schizandrin reference substance solution, need testing solution, press the text total lignan and measure item method suggested down, in 200～400nm wavelength 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 lignan content in the spectrophotometry shengmai injection.
Schizandrin reference substance 15mg is got in the preparation of 3 reference substance solution, puts in the 50ml measuring bottle, adds an amount of sonicated of methanol solution (power 250W, frequency 33KHz) makes dissolving, take out, put to room temperature, add methanol solution to scale, shake up, promptly get (containing schizandrin 0.3mg among every 1ml).
The preparation precision of 4 typical 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, add methyl alcohol to scale, shaking up, is blank with the retinue solvent, according to spectrophotometric method (appendix VB of Chinese Pharmacopoeia version in 2000), measure absorbance log at 250nm wavelength place, (μ g/ml) is horizontal ordinate with concentration, and absorbance log is that ordinate is figure, the drawing standard curve.
Total lignan standard curve determination result
| Numbering | Schizandrin concentration (μ g/ml) | Absorbance log |
| 1 | 2.908 | 0.118 |
| 2 | 5.816 | 0.245 |
| 3 | 11.632 | 0.491 |
| 4 | 17.448 | 0.732 |
| 5 | 23.264 | 0.971 |
| 6 | 29.080 | 1.217 |
Regression equation: Y=0.0418x+0.0003;
Related coefficient: γ=0.9999;
The result shows: schizandrin is good in 2.908～29.080 μ g/ml scope internal linear.
As calculated, the typical curve of schizandrin is crossed initial point, therefore selects for use one point external standard method to measure the content of total lignan in the pulse restoring injection.
The accurate schizandrin reference substance solution (concentration: 0.2908mg/ml) 0.6ml of drawing of 5 precision test, put in the 10ml measuring bottle, add methyl alcohol to scale, shake up, with the retinue solvent is blank, according to spectrophotometric method (an appendix V of Chinese Pharmacopoeia version in 2000 B), measure absorbance log, replication 5 times at 250nm wavelength place.
The precision test
| Numbering | 1 | 2 | 3 | 4 | 5 | Average | RSD(％) |
| Absorbance log | 0.732 | 0.733 | 0.731 | 0.734 | 0.732 | 0.732 | 0.16 |
The result shows that reference substance solution precision is good.
6 stability tests
6.1 the accurate schizandrin reference substance solution (concentration: 0.2908mg/ml) 0.6ml of drawing of reference substance solution stability test, put in the 10ml measuring bottle, add methyl alcohol to scale, shake up, with the retinue solvent is blank, according to spectrophotometric method (an appendix V of Chinese Pharmacopoeia version in 2000 B), measure absorbance log at 250nm wavelength place, respectively once at 0,2,4,8,12 hour replication.
The reference substance solution stability test
| Time (h) | 0 | 2 | 4 | 8 | 12 | Average | RSD(％) |
| Absorbance log | 0.732 | 0.733 | 0.731 | 0.734 | 0.732 | 0.732 | 0.16 |
The result shows that reference substance solution has good stability.
6.2 need testing solution stability test precision is measured shengmai injection 4ml, puts in the 50ml measuring bottle, adds an amount of sonicated of methanol solution (power 250W, frequency 33KHz) makes dissolving, take out, put to room temperature, be diluted to scale with methanol solution, shaking up, filter, is blank with the retinue solvent, according to spectrophotometric method (an appendix V of Chinese Pharmacopoeia version in 2000 B), measure absorbance log at 250nm wavelength place, calculate with one point external standard method, respectively once at 0,2,4,8,12 hour replication
The need testing solution stability test
| Time (h) | 0 | 2 | 4 | 8 | 12 | Average | RSD(％) |
| Content (mg/ props up) | 2.03 | 2.05 | 2.02 | 2.04 | 2.03 | 2.03 | 0.56 |
The result shows that need testing solution is good at the 30min internal stability.
7 replica test precisions are measured shengmai injection 4ml (totally 5 parts), split in the 50ml measuring bottle, add an amount of sonicated of methanol solution (power 250W, frequency 33KHz) makes dissolving, take out, put to room temperature, be diluted to scale with methanol solution, shaking up, filter, is blank with the retinue solvent, according to spectrophotometric method (an appendix V of Chinese Pharmacopoeia version in 2000 B), measure absorbance log at 250nm wavelength place, calculate with one point external standard method, promptly.
Replica test
| Numbering | 1 | 2 | 3 | 4 | 5 | Average | RSD(％) |
| Content (mg/ props up) | 2.06 | 2.01 | 2.04 | 2.06 | 2.05 | 2.04 | 1.01 |
The result shows that repeatability is good.
The application of sample absorption method is adopted in the test of 8 average recoveries, and precision is measured shengmai injection 2ml (totally 5 parts), and accurate the title decides, split in the 50ml measuring bottle, precision is measured reference substance solution (C=0.2722mg/ml) 1.5ml, splits in the above-mentioned 50ml measuring bottle, add an amount of sonicated of methanol solution (power 250W, frequency 33KHz) and make dissolving, take out, put to room temperature, be diluted to scale, shake up with methanol solution, filter, wavelength place at 250nm measures absorbance log, calculates with one point external standard method, promptly.
The content of total lignan is in the shengmai injection: 2.04mg/ props up.
The average recovery test
Average recovery rate=97.91%, RSD=0.95%.
The assay of 9 samples is got this product three batch samples, according to the preparation of text need testing solution and the operation under the determination method item, working sample content.
The assay of total lignan
| Lot number | Total lignan content (mg/ props up) |
| 1 | 2.09 |
| 2 | 2.07 |
| 3 | 2.01 |
From above test as can be known, it is good to adopt the method for the invention to measure the content precision of total lignan in the pulse restoring injection, stability and repeatability, recovery height, and method is easy, and data are accurate, reliable.
The content assaying method of total polysaccharides in experimental example 16 pulse restoring injections
Monose 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose (C of 9.008mg
6H
12O
6), convert out the content of every bottle of monose thus.
Total reducing 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose (C of 9.008mg
6H
12O
6), calculate sugar contents in every bottle in the sample with this.
The typical curve precision takes by weighing 105 ℃ of anhydrous dextrose 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose (C of 9.008mg
6H
12O
6).Consuming vs volume (ml) with sample is horizontal ordinate, is that ordinate is figure with the amount (mg) of anhydrous dextrose, the drawing standard curve.
The anhydrous dextrose typical curve
| Numbering | The sample vs consumes volume (ml) | Anhydrous dextrose (mg) |
| 1 | 5.65 | 50.24 |
| 2 | 11.25 | 101.87 |
| 3 | 16.85 | 149.58 |
| 4 | 22.65 | 201.36 |
| 5 | 28.25 | 252.81 |
| 6 | 33.74 | 300.79 |
Regression equation: Y=8.9093x+0.3139;
Related coefficient: γ=0.9999;
The result shows: anhydrous dextrose is good in 50.24～300.79 μ g scope internal linear.
As calculated, the typical curve of anhydrous dextrose is crossed initial point, therefore selects for use one point external standard method to measure the content of total polysaccharides in the pulse restoring injection.
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 determination method item, measures, promptly.
Repeated experiment
| Numbering | Weighing (mg) | Total reducing sugar (mg/ bottle) | Monose (mg/ bottle) | Total polysaccharides (mg/ bottle) |
| 1 | 470.54 | 172.65 | 85.64 | 87.01 |
| 2 | 502.03 | 177.84 | 89.21 | 88.63 |
| 3 | 512.39 | 171.29 | 86.37 | 84.92 |
| 4 | 502.47 | 176.01 | 88.59 | 87.42 |
| 5 | 508.61 | 174.39 | 86.87 | 87.52 |
| Average | Zhao | 174.44 | 87.34 | 87.10 |
| RSD(％) | Zhao | 1.49 | 1.73 | 1.56 |
The application of sample absorption method is adopted in recovery experiment, gets this product under the content uniformity item, gets content, and mixing is therefrom got about 250mg (6 parts), splits in the iodine flask; Be taken at 105 ℃ of about 35mg of anhydrous dextrose 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 vs immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank test, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose (C of 9.008mg
6H
12O
6).
Pulse restoring injection contents of monosaccharides: 87.34mg/ bottle.
The test of anhydrous dextrose average recovery
| Numbering | Test sample weighing (mg) | Monose amount (mg) in the test sample | Anhydrous dextrose addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 244.86 | 35.54 | 34.92 | 70.12 | 99.03 |
| 2 | 250.17 | 36.31 | 37.26 | 72.91 | 98.23 |
| 3 | 239.64 | 34.79 | 35.84 | 70.33 | 99.16 |
| 4 | 255.73 | 37.12 | 36.11 | 71.94 | 96.43 |
| 5 | 260.92 | 37.87 | 35.58 | 72.93 | 98.54 |
| 6 | 251.53 | 36.51 | 34.79 | 70.82 | 98.62 |
Average recovery rate=98.34%; RSD=1.01%.
The assay of sample is got in this product three batches of test agents, presses the preparation and the operation down of determination method item of text need testing solution.
Total polysaccharides assay result
| Lot number | Total polysaccharides content (mg/ bottle) |
| 1 | 84.93 |
| 2 | 90.22 |
| 3 | 87.65 |
From above test as can be known, it is good to adopt the method for the invention to measure the content precision of total polysaccharides in the pulse restoring injection, stability and repeatability, recovery height, and method is easy, and data are accurate, reliable.
Ophiopogonin B in experimental example 17 pulse restoring injections, ophiopogonin D, ophiopogonin D ' content assaying method
1. instrument and reagent
(1) instrument: SHIMADZU 2010Aht high performance liquid chromatograph; The TU-1810SPC ultraviolet spectrophotometer; SARTORIUS BP211D electronic analytical balance.
(2) reagent: ophiopogonin B, ophiopogonin D, ophiopogonin D '
2. it is an amount of that the selection precision that detects wavelength takes by weighing ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adds methyl alcohol respectively and make the solution that every 1ml contains 0.1mg, scans in the 200-400nm wavelength coverage respectively.The result shows: ophiopogonin B, ophiopogonin D, ophiopogonin D ' all absorption maximum is arranged at the 203nm place, therefore select the detection wavelength of 203nm as assay.
3. chromatographic condition: Dikma ODS (4.6 * 250mm, 5um); Moving phase: acetonitrile-water (90: 10) is a moving phase; The detection wavelength is 203nm; Flow velocity: 1.0ml/min.
4. system suitability test
4.1 it is an amount of that the preparation precision of reference substance solution takes by weighing ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adding methyl alcohol makes every 1ml and contains ophiopogonin B 0.007mg, ophiopogonin D 0.007mg, the reference substance mixed solution of ophiopogonin D ' 0.002mg, promptly.
4.2 the preparation of need testing solution is got shengmai injection as need testing solution.
4.3 the preparation of negative need testing solution takes by weighing the negative sample that does not contain the tuber of dwarf lilyturf, according to preparation method's preparation of need testing solution, as negative need testing solution.
Accurate respectively reference substance solution, need testing solution, each 10 μ l of negative sample solution of drawing inject liquid chromatograph, measure the record chromatogram.As seen from the figure, ophiopogonin B in the test sample, ophiopogonin D, ophiopogonin D ' peak separate good, and it is clear to separate with close peak, and fully, ophiopogonin B, ophiopogonin D in the negative test sample, ophiopogonin D ' peak is noiseless.
5. linear relationship is investigated precision and is taken by weighing ophiopogonin B reference substance 10.14mg, ophiopogonin D reference substance 10.06mg, ophiopogonin D ' reference substance 6.59mg, split in the 50ml measuring bottle, add methyl alcohol respectively and make dissolving and fixed to scale in right amount, shake up, precision is measured ophiopogonin B reference substance solution 0.7ml, ophiopogonin D reference substance solution 0.7ml, ophiopogonin D ' reference substance solution 0.4ml is put in the 10ml measuring bottle altogether, and it is fixed to scale to add methyl alcohol, shake up, the accurate 2 μ l that draw, 4 μ l, 6 μ l, 8 μ l, 10 μ l inject liquid chromatograph, are ordinate with the peak area, sample size (μ g) is figure for horizontal ordinate, the drawing standard curve.
The ophiopogonin B linear relationship is investigated
| Numbering | Ophiopogonin B (μ g) | Peak area |
| 1 | 0.028392 | 21894 |
| 2 | 0.056784 | 43805 |
| 3 | 0.085176 | 66601 |
| 4 | 0.113568 | 88329 |
| 5 | 0.141960 | 109588 |
Regression equation: Y=774556.21X+69.80;
The coefficient of determination: γ=0.9999;
Ophiopogonin B is good in 0.028392～0.14196 μ g scope internal linear relation.
The ophiopogonin D linear relationship is investigated
| Numbering | Ophiopogonin D (μ g) | Peak area |
| 1 | 0.028168 | 18411 |
| 2 | 0.056336 | 36824 |
| 3 | 0.084504 | 55119 |
| 4 | 0.112672 | 73802 |
| 5 | 0.140840 | 92108 |
Regression equation: Y=654544.16X-58.80;
The coefficient of determination: γ=0.9999;
Ophiopogonin D is good in 0.028168～0.14084 μ g scope internal linear relation.
Ophiopogonin D ' linear relationship is investigated
| Numbering | Ophiopogonin D ' (μ g) | Peak area |
| 1 | 0.010544 | 18859 |
| 2 | 0.021088 | 37804 |
| 3 | 0.031632 | 56821 |
| 4 | 0.042176 | 75621 |
| 5 | 0.052720 | 94338 |
Regression equation: Y=1790354.70X+56.10;
The coefficient of determination: γ=0.9999;
Ophiopogonin D ' good in 0.010544～0.052720 μ g scope internal linear relation.
6. precision test precision is drawn reference substance mixed solution (every 1ml contains ophiopogonin B 8.112 μ g respectively, ophiopogonin D 8.048 μ g, ophiopogonin D ' 2.636 μ g) 10 μ l, inject liquid chromatograph, the record peak area, METHOD FOR CONTINUOUS DETERMINATION 5 times, the precision of investigation reference substance solution.
The test of reference substance precision
| Test number (TN) | 1 | 2 | 3 | 4 | 5 | Average | RSD(％) |
| Ophiopogonin B | 63356 | 64187 | 62195 | 63314 | 62868 | 63184 | 1.16 |
| Ophiopogonin D | 51196 | 52137 | 53268 | 52571 | 53094 | 52453 | 1.59 |
| Ophiopogonin D ' | 47615 | 45508 | 46309 | 47703 | 46925 | 46672 | 1.71 |
The result shows that reference substance solution precision is good.
7. the accurate shengmai injection 10 μ l that draw of stability test inject liquid chromatograph, calculate with one point external standard method, that is, measure at 0,6,12,24,48 hour sample introduction respectively.
The reference substance solution stability test
| Time (h) | 0 | 6 | 12 | 24 | 48 | Average | RSD(％) |
| Ophiopogonin B (mg/ props up) | 0.0814 | 0.0822 | 0.0813 | 0.0807 | 0.0842 | 0.0820 | 1.66 |
| Ophiopogonin D (mg/ props up) | 0.0818 | 0.0805 | 0.0834 | 0.0816 | 0.0822 | 0.0819 | 1.28 |
| Ophiopogonin D ' (mg/ props up) | 0.0203 | 0.0205 | 0.0206 | 0.0201 | 0.0208 | 0.0205 | 1.32 |
The result shows that reference substance solution has good stability.
8. the accurate shengmai injection 10 μ l that draw of replica test inject liquid chromatograph, calculate with one point external standard method, promptly.
Replica test
| Numbering | 1 | 2 | 3 | 4 | 5 | Average | RSD(％) |
| Ophiopogonin B (mg/ props up) | 0.0827 | 0.0834 | 0.0818 | 0.0825 | 0.0836 | 0.0828 | 0.88 |
| Ophiopogonin D (mg/ props up) | 0.0821 | 0.0831 | 0.0827 | 0.0826 | 0.0818 | 0.0825 | 0.62 |
| Ophiopogonin D ' (mg/ props up) | 0.0211 | 0.0207 | 0.0205 | 0.0214 | 0.0208 | 0.0209 | 1.69 |
The result shows that repeatability is good.
9. the application of sample absorption method is adopted in the average recovery test, and precision is measured shengmai injection 5ml, splits in the 10ml measuring bottle; Precision is measured the reference substance mixed solution, and (every 1ml contains ophiopogonin B 0.03728mg respectively, ophiopogonin D 0.03564mg, the 1ml (totally 5 parts) of ophiopogonin D ' 0.00928mg), split in the above-mentioned 10ml measuring bottle, add an amount of sonicated of methyl alcohol (power 250W, frequency 33KHz) 10 minutes, take out, put, add methyl alcohol to scale to room temperature, shake up, filter, the accurate subsequent filtrate 10 μ l that draw inject liquid chromatograph, calculate with one point external standard method, promptly.
Ophiopogonin B content in the shengmai injection: 0.0828mg/ props up;
Ophiopogonin D content in the shengmai injection: 0.0825mg/ props up;
Ophiopogonin D ' content in the shengmai injection: 0.0209mg/ props up.
The test of ophiopogonin B average recovery
| Numbering | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 0.0414 | 0.03728 | 0.07798 | 98.12 |
| 2 | 0.0414 | 0.03728 | 0.07832 | 99.04 |
| 3 | 0.0414 | 0.03728 | 0.07777 | 97.56 |
| 4 | 0.0414 | 0.03728 | 0.07809 | 98.41 |
| 5 | 0.0414 | 0.03728 | 0.07840 | 99.25 |
Ophiopogonin B average recovery rate=98.48%; RSD=0.70%.
The test of ophiopogonin D average recovery
| Numbering | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 0.04125 | 0.03564 | 0.07608 | 97.72 |
| 2 | 0.04125 | 0.03564 | 0.07623 | 98.15 |
| 3 | 0.04125 | 0.03564 | 0.07673 | 99.56 |
| 4 | 0.04125 | 0.03564 | 0.07631 | 98.37 |
| 5 | 0.04125 | 0.03564 | 0.07654 | 99.03 |
Ophiopogonin D average recovery rate=98.57%; RSD=0.74%.
Ophiopogonin D ' average recovery test
| Numbering | Pure product amount (mg) in the test sample | Pure product add (mg) | Measured value (mg) | The recovery (%) |
| 1 | 0.01045 | 0.00928 | 0.01965 | 99.15 |
| 2 | 0.01045 | 0.00928 | 0.01966 | 99.26 |
| 3 | 0.01045 | 0.00928 | 0.01934 | 95.84 |
| 4 | 0.01045 | 0.00928 | 0.01945 | 97.03 |
| 5 | 0.01045 | 0.00928 | 0.01964 | 99.08 |
Ophiopogonin D ' average recovery rate=98.07%; RSD=1.58%.
10. three batches of pilot scale sample determinations
Get three batches in this product sample, press the described method of text and handle.
Three batch sample assay results
| Lot number | 1 | 2 | 3 |
| Ophiopogonin B (mg/ props up) | 0.0834 | 0.0819 | 0.0839 |
| Ophiopogonin D (mg/ props up) | 0.0821 | 0.0816 | 0.0824 |
| Ophiopogonin D ' (mg/ props up) | 0.0232 | 0.0235 | 0.0243 |
From above test as can be known, adopt the method for the invention measure ophiopogonin B in the shengmai injection, ophiopogonin D, ophiopogonin D ' content precision, stability and repeatability good, recovery height, method is easy, data are accurately, reliably.
The content assaying method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in experimental example 18 pulse restoring injections
1. instrument and reagent
(1) instrument: SHIMADZU 2010Aht high performance liquid chromatograph; The TU-1810SPC ultraviolet spectrophotometer; SARTORIUS BP211D electronic analytical balance.
(2) reagent: Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin
2. it is an amount of that the selection precision that detects wavelength takes by weighing Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adds methyl alcohol respectively and make the solution that every 1ml contains 0.1mg, scans in the 200-400nm wavelength coverage respectively.The result shows: Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin all have absorption maximum at the 203nm place, therefore select the detection wavelength of 203nm as assay.
3. chromatographic condition: Dikma ODS (4.6 * 250mm, 5um); Moving phase: acetonitrile-water (90: 10) is a moving phase; The detection wavelength is 203nm; Flow velocity: 1.0ml/min.
4. system suitability test
4.1 it is an amount of that the preparation precision of reference substance solution takes by weighing Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adding methyl alcohol makes every 1ml and contains Ruscus aculeatus L. sapogenin 0.002mg, diosgenin 0.002mg, the reference substance mixed solution of ruscogenin 0.002mg, promptly.
4.2 the preparation precision of need testing solution is measured shengmai injection 10ml, puts in the round-bottomed flask, adds 3% sulfuric acid 10ml and refluxes 4 hours, take out, put, transfer pH to neutral with 3% sodium hydroxide solution to room temperature, extract 3 times 15ml and also extract at every turn with methenyl choloride, evaporate to dryness, residue makes dissolving in right amount with methyl alcohol and quantitatively is transferred in the 10ml measuring bottle, and it is fixed to scale to add methyl alcohol, shakes up, filter with miillpore filter, get subsequent filtrate promptly.
4.3 the preparation of negative need testing solution takes by weighing the negative sample that does not contain the tuber of dwarf lilyturf, according to preparation method's preparation of need testing solution, as negative need testing solution.
Accurate respectively reference substance solution, need testing solution, each 10 μ l of negative sample solution of drawing inject liquid chromatograph, measure the record chromatogram.As seen from the figure, Ruscus aculeatus L. sapogenin in the test sample, diosgenin, ruscogenin peak separate good, and it is clear to separate with close peak, and fully, Ruscus aculeatus L. sapogenin, diosgenin in the negative test sample, the ruscogenin peak is noiseless.
5. linear relationship is investigated precision and is taken by weighing Ruscus aculeatus L. sapogenin reference substance 6.71mg, diosgenin reference substance 6.94mg, ruscogenin reference substance 6.63mg, put altogether in the 50ml measuring bottle, add methyl alcohol and make dissolving and fixed in right amount to scale, shake up, precision is measured 0.4ml, puts in the 10ml measuring bottle, and it is fixed to scale to add methyl alcohol, shake up, accurate 2 μ l, 4 μ l, 6 μ l, 8 μ l, the 10 μ l of drawing inject liquid chromatograph, are ordinate with the peak area, sample size (μ g) is figure for horizontal ordinate, the drawing standard curve.
The Ruscus aculeatus L. sapogenin linear relationship is investigated
| Numbering | Ruscus aculeatus L. sapogenin (μ g) | Peak area |
| 1 | 0.010736 | 15729 |
| 2 | 0.021472 | 31526 |
| 3 | 0.032208 | 47338 |
| 4 | 0.042944 | 62952 |
| 5 | 0.053680 | 78903 |
Regression equation: Y=1469578.99X-42.60;
The coefficient of determination: γ=0.9999;
Ruscus aculeatus L. sapogenin is good in 0.010736～0.053680 μ g scope internal linear relation.
The diosgenin linear relationship is investigated
| Numbering | Diosgenin (μ g) | Peak area |
| 1 | 0.011104 | 16593 |
| 2 | 0.022208 | 33041 |
| 3 | 0.033312 | 49825 |
| 4 | 0.044416 | 66407 |
| 5 | 0.055520 | 82831 |
Regression equation: Y=1493533.86X-13.20;
The coefficient of determination: γ=0.9999;
Diosgenin is good in 0.011104～0.055520 μ g scope internal linear relation.
The ruscogenin linear relationship is investigated
| Numbering | Ruscogenin (μ g) | Peak area |
| 1 | 0.010608 | 20521 |
| 2 | 0.021216 | 41137 |
| 3 | 0.031824 | 61459 |
| 4 | 0.042432 | 82003 |
| 5 | 0.053040 | 102711 |
Regression equation: Y=1934822.78X-7.60;
The coefficient of determination: γ=0.9999;
Ruscogenin is good in 0.010608～0.053040 μ g scope internal linear relation.
6. precision test precision is drawn reference substance mixed solution (every 1ml contains Ruscus aculeatus L. sapogenin 2.684 μ g respectively, diosgenin 2.776 μ g, ruscogenin 2.652 μ g) 10 μ l, inject liquid chromatograph, the record peak area, METHOD FOR CONTINUOUS DETERMINATION 5 times, the precision of investigation reference substance solution.
The test of reference substance precision
| Test number (TN) | 1 | 2 | 3 | 4 | 5 | Average | RSD(％) |
| Ruscus aculeatus L. sapogenin | 39429 | 40107 | 41135 | 40863 | 40526 | 40412 | 1.66 |
| Diosgenin | 41137 | 40928 | 42056 | 40764 | 41963 | 41370 | 1.45 |
| Ruscogenin | 50847 | 51093 | 50625 | 51147 | 52036 | 51150 | 1.05 |
The result shows that reference substance solution precision is good.
7. stability test precision is measured shengmai injection 10ml, put in the round-bottomed flask, add 3% sulfuric acid 10ml and refluxed 4 hours, take out, put to room temperature, transfer pH to neutral with 3% sodium hydroxide solution, extract 3 times 15ml and also extract at every turn with methenyl choloride, evaporate to dryness, residue makes dissolving in right amount with methyl alcohol and quantitatively is transferred in the 10ml measuring bottle, and it is fixed to scale to add methyl alcohol, shakes up, filter, the accurate 10 μ l that draw inject liquid chromatograph, calculate with one point external standard method, that is, measure at 0,6,12,24,48 hour sample introduction respectively.
The reference substance solution stability test
| Time (h) | 0 | 6 | 12 | 24 | 48 | Average | RSD(％) |
| Ruscus aculeatus L. sapogenin (mg/ props up) | 0.021 4 | 0.0205 | 0.0211 | 0.0208 | 0.0206 | 0.0209 | 1.77 |
| Diosgenin (mg/ props up) | 0.020 3 | 0.0199 | 0.0205 | 0.0207 | 0.0201 | 0.0203 | 1.56 |
| Ruscogenin (mg/ props up) | 0.021 4 | 0.0217 | 0.0223 | 0.0221 | 0.0219 | 0.0219 | 1.60 |
The result shows that reference substance solution has good stability.
8. the replica test precision is measured shengmai injection 10ml (totally 5 parts), the accurate title, decide, and splits in the round-bottomed flask, adds 3% sulfuric acid 10ml and refluxed 4 hours, take out, put to room temperature, transfer pH to neutral, extract 3 times with methenyl choloride with 3% sodium hydroxide solution, each 15ml, with and extract, evaporate to dryness, residue with methyl alcohol make in right amount the dissolving and quantitatively be transferred in the 10ml measuring bottle, it is fixed to scale to add methyl alcohol, shake up, filter, the accurate 10 μ l that draw, the accurate 10 μ l that draw, inject liquid chromatograph, calculate with one point external standard method, promptly.
Replica test
| Numbering | 1 | 2 | 3 | 4 | 5 | Average | RSD(％) |
| Ruscus aculeatus L. sapogenin (mg/ props up) | 0.0223 | 0.0221 | 0.0226 | 0.0218 | 0.0225 | 0.0223 | 1.44 |
| Diosgenin (mg/ props up) | 0.0214 | 0.0209 | 0.0211 | 0.0207 | 0.0204 | 0.0209 | 1.82 |
| Ruscogenin (mg/ props up) | 0.0226 | 0.0227 | 0.0229 | 0.0224 | 0.0225 | 0.0226 | 0.85 |
The result shows that repeatability is good.
9. the application of sample absorption method is adopted in the average recovery test, and precision is measured shengmai injection 5ml (totally 5 parts), splits in the round-bottomed flask; Precision is measured the reference substance mixed solution, and (every 1ml contains Ruscus aculeatus L. sapogenin 0.00904mg respectively, diosgenin 0.00948mg, ruscogenin 0.00848mg) 1ml (totally 5 parts), split in the above-mentioned round-bottomed flask, adding 3% sulfuric acid 10ml respectively refluxed 4 hours, take out, put, transfer pH to neutral with 3% sodium hydroxide solution to room temperature, extract 3 times with methenyl choloride, 15ml and also extract at every turn, evaporate to dryness, residue makes dissolving in right amount with methyl alcohol and quantitatively is transferred in the 10ml measuring bottle, add methyl alcohol and decide to shake up, filter to scale, the accurate subsequent filtrate 10 μ l that draw, inject liquid chromatograph, calculate with one point external standard method, promptly.
Ruscus aculeatus L. sapogenin content in the shengmai injection: 0.0223mg/ props up;
Diosgenin content in the shengmai injection: 0.0209mg/ props up;
Ruscogenin content in the shengmai injection: 0.0226mg/ props up.
The test of Ruscus aculeatus L. sapogenin average recovery
| Numbering | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 0.01115 | 0.00904 | 0.01994 | 97.25 |
| 2 | 0.01115 | 0.00904 | 0.02007 | 98.63 |
| 3 | 0.01115 | 0.00904 | 0.02011 | 99.06 |
| 4 | 0.01115 | 0.00904 | 0.01987 | 96.42 |
| 5 | 0.01115 | 0.00904 | 0.01993 | 97.15 |
Ruscus aculeatus L. sapogenin average recovery rate=97.70%; RSD=1.13%.
The test of diosgenin average recovery
Diosgenin average recovery rate=98.22%; RSD=1.06%.
The test of ruscogenin average recovery
| Numbering | Pure product amount (mg) in the test sample | Pure product addition (mg) | Measured value (mg) | The recovery (%) |
| 1 | 0.0113 | 0.00848 | 0.01970 | 99.05 |
| 2 | 0.0113 | 0.00848 | 0.01962 | 98.16 |
| 3 | 0.0113 | 0.00848 | 0.01964 | 98.34 |
| 4 | 0.0113 | 0.00848 | 0.01953 | 97.06 |
| 5 | 0.0113 | 0.00848 | 0.01948 | 96.47 |
Ruscogenin average recovery rate=97.82%; RSD=1.06%.
10. three batches of pilot scale sample determinations
Get three batches in this product sample, press the described method of text and handle.
Three batch sample assay results
| Lot number | 1 | 2 | 3 |
| Ruscus aculeatus L. sapogenin (mg/ props up) | 0.0221 | 0.0246 | 0.0239 |
| Diosgenin (mg/ props up) | 0.0226 | 0.0218 | 0.0242 |
| Ruscogenin (mg/ props up) | 0.0236 | 0.0229 | 0.0241 |
From above test as can be known, it is good to adopt the method for the invention to measure the content precision of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the shengmai injection, stability and repeatability, recovery height, and method is easy, and data are accurate, reliable.
Embodiment
Embodiment 1: adopt liquid chromatography for measuring based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics finger-print:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds water and make the solution that every 1ml contains 50mg, with the miillpore filter 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 methyl alcohol 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 is 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) formulation of standard finger-print: with said method as formulate based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 11;
(5) based on the described method in (1)～(3) as red ginseng or genseng in the freeze-dried powder to be measured and the tuber of dwarf lilyturf composition characteristics the means of testing of finger-print, the finger-print of preparation testing sample;
(6) with the contrast of freeze-dried powder finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;
II. in the freeze-dried powder finger-print to be measured, non-total peak area is no more than 5% of total peak area;
III., the odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the test sample finger-print, its difference is not more than ± 30%.
Embodiment 2: adopt the finger-print of liquid chromatography for measuring based on fruit of Chinese magnoliavine composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds water and make the solution that every 1ml contains 50mg, with the miillpore filter 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 schizandrin 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; Moving phase is methanol-water, gradient elution, solvent ratios are from 0 minute to 6 minutes, and the ratio of methyl alcohol is 68%, from 6 minutes to 8 minutes, the ratio of methyl alcohol rises to 76% by 68%, and from 8 minutes to 10 minutes, the ratio of methyl alcohol was 76%, from 10 minutes to 15 minutes, the ratio of methyl alcohol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methyl alcohol was 68%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;
(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of fruit of Chinese magnoliavine composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 17
(5) based on the means of testing of the described method in (1)～(3) as the finger-print of freeze-dried powder fruit of Chinese magnoliavine composition characteristics to be measured, the finger-print of preparation testing sample;
(6) with the contrast of freeze-dried powder finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;
II. in the freeze-dried powder finger-print to be measured, non-total peak area is no more than 5% of total peak area;
The odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the freeze-dried powder finger-print III. to be measured, its difference is not more than ± 30%.
Embodiment 3: adopt liquid chromatography for measuring based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics finger-print:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds methyl alcohol and make the solution that every 1ml contains 50mg, with the miillpore filter filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;
(2) preparation of object of reference solution: precision takes by weighing ginsenoside Rb
1In right amount, add ethanol 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 sodium dihydrogen phosphate, and Mobile phase B is acetonitrile-water 85: 15, gradient elution, solvent ratios is from 0 minute to 4 minutes, the ratio of Mobile phase B is 22%, and from 4 minutes to 40 minutes, the ratio of Mobile phase B rose to 60% by 22%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 60%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 77% by 60%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 22% by 77%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;
(4) formulation of standard finger-print: with said method as formulate based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 13;
(5) based on the described method in (1)～(3) as red ginseng or genseng in the freeze-dried powder to be measured and the tuber of dwarf lilyturf composition characteristics the means of testing of finger-print, the finger-print of preparation testing sample;
(6) with the contrast of freeze-dried powder finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;
II. in the freeze-dried powder finger-print to be measured, non-total peak area is no more than 5% of total peak area;
The odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the freeze-dried powder finger-print III. to be measured, its difference is not more than ± 30%.
Embodiment 4: adopt the finger-print of liquid chromatography for measuring based on fruit of Chinese magnoliavine composition characteristics:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds methyl alcohol and make the solution that every 1ml contains 50mg, with the miillpore filter 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 wuweizi alcohol B reference substance, adds methyl alcohol 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; Moving phase is acetonitrile-0.1% phosphoric acid, gradient elution, solvent ratios are from 0 minute to 5 minutes, and the ratio of acetonitrile is 60%, from 5 minutes to 10 minutes, the ratio of acetonitrile rises to 70% by 60%, and from 10 minutes to 12 minutes, the ratio of acetonitrile was 70%, from 12 minutes to 20 minutes, the ratio of acetonitrile reduces to 62% by 70%, and from 12 minutes to 60 minutes, the ratio of acetonitrile was 62%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;
(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of fruit of Chinese magnoliavine composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 20
(5) based on the means of testing of the described method in (1)～(3) as the finger-print of fruit of Chinese magnoliavine composition characteristics in the freeze-dried powder to be measured, the finger-print of preparation testing sample;
(6) with the contrast of freeze-dried powder finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;
II. in the freeze-dried powder finger-print to be measured, non-total peak area is no more than 5% of total peak area;
The odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the freeze-dried powder finger-print III. to be measured, its difference is not more than ± 30%.
Embodiment 5: adopt liquid chromatography for measuring based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics finger-print:
(1) preparation of need testing solution: get parenteral solution as need testing solution;
(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the ginsenoside Rf, adds ethanol 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 1% glacial acetic acid aqueous solution, Mobile phase B is methanol-water 95: 5, gradient elution, solvent ratios are from 0 minute to 10 minutes, and the ratio of Mobile phase B is 30%, from 10 minutes to 35 minutes, the ratio of Mobile phase B rises to 85% by 30%, and from 35 minutes to 55 minutes, the ratio of Mobile phase B was 85%, from 55 minutes to 70 minutes, the ratio of Mobile phase B reduced to 30% by 85%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;
(4) formulation of standard finger-print: with said method as formulate based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 15;
(5) based on the described method in (1)～(3) as red ginseng or genseng in the parenteral solution to be measured and the tuber of dwarf lilyturf composition characteristics the means of testing of finger-print, the finger-print of preparation testing sample;
(6) with the contrast of parenteral solution finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of parenteral solution to be measured, should be 0.90～1.00;
II. in the parenteral solution finger-print to be measured, non-total peak area is no more than 5% of total peak area;
The odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the parenteral solution finger-print III. to be measured, its difference is not more than ± 30%.
Embodiment 6: adopt the finger-print of liquid chromatography for measuring based on fruit of Chinese magnoliavine composition characteristics:
(1) preparation of need testing solution: get parenteral solution as need testing solution;
(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the schizandrin A reference substance, adds ethanol 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; Moving phase is acetonitrile-1% glacial acetic acid aqueous solution, gradient elution, solvent ratios is from 0 minute to 5 minutes, the ratio of acetonitrile is 60%, and from 5 minutes to 10 minutes, the ratio of acetonitrile rose to 70% by 60%, from 10 minutes to 15 minutes, the ratio of acetonitrile is 70%, and from 15 minutes to 60 minutes, the ratio of acetonitrile reduced to 54% by 70%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;
(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of fruit of Chinese magnoliavine composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 14
(5) based on the means of testing of the described method in (1)～(3) as the finger-print of fruit of Chinese magnoliavine composition characteristics in the parenteral solution to be measured, the finger-print of preparation testing sample;
(6) with the contrast of parenteral solution finger-print to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:
I. calculate the similarity of the finger-print and the standard finger-print of parenteral solution to be measured, should be 0.90～1.00;
II. in the parenteral solution finger-print to be measured, non-total peak area is no more than 5% of total peak area;
The odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the parenteral solution finger-print III. to be measured, its difference is not more than 30%.
Embodiment 7: the thin-layer chromatography discrimination method of the fruit of Chinese magnoliavine, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, adds methenyl choloride and extracts, and filters, and filtrate volatilizes, and residue adds the methenyl choloride dissolving, as need testing solution; The preparation of fruit of Chinese magnoliavine control medicinal material solution: it is an amount of to get fruit of Chinese magnoliavine control medicinal material, adds methenyl choloride and extracts, and filters, and filtrate volatilizes, and residue adds methenyl choloride dissolving, medicinal material solution in contrast; The preparation of reference substance solution: get schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B reference substance, add methenyl choloride respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be developping agent, launch with sherwood oil (30～60 ℃)-upper solution of 15: 5: 1 of ethyl formate-formic acid, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 8: the liquid chromatography of schizandrin, schizandrin A, deoxyschizandrin is differentiated in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with schizandrin, schizandrin A, deoxyschizandrin reference substance is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methanol-water is a moving phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 9: the thin-layer chromatography discrimination method of the tuber of dwarf lilyturf in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, adds 7: 3 mixed solutions of methenyl choloride-methyl alcohol and extracts, and filters, and filtrate volatilizes, and residue adds the methenyl choloride dissolving, as need testing solution; Other gets the control medicinal material tuber of dwarf lilyturf, shines medicinal material solution in pairs with legal system; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be at 80: 5: 0.1 developping agent, launch, take out, dry, put under the uviol lamp 254nm and inspect with toluene-methyl alcohol-glacial acetic acid, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color, negative noiseless;
Embodiment 10: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' the thin-layer chromatography discrimination method:
It is an amount of to get freeze-dried powder to be measured, and extracting n-butyl alcohol is used in water dissolving back, and extract volatilizes, and the residue dissolve with methanol is as need testing solution; Other gets ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adds methyl alcohol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developping agent with ethyl acetate-methanol-water at 15: 5: 1, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ dry by the fire to spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 11: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' liquid chromatography differentiate:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopts liquid phase chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-water is a moving phase at 90: 10, and the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 12: the thin-layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, adds 3% sulphuric acid hydrolysis 4 hours, takes out, and puts to room temperature, regulates pH to neutral, filters, and filtrate volatilizes, and residue dissolves with methenyl choloride, as need testing solution; Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adds methenyl choloride respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, be developping agent with normal hexane-ethyl acetate-water at 1: 1: 1, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 90 ℃ dry by the fire to spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 13: the liquid chromatography of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the round-bottomed flask, and refluxing 4 hours with 3% sulfuric acid in the back that is dissolved in water, takes out, put to room temperature, transfer pH to neutral, extract with methenyl choloride, extract volatilizes, the residue dissolve with methanol filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-water is a moving phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 14: red ginseng or genseng, ginsenoside Rg in the freeze-dried powder
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin-layer chromatography discrimination method:
It is an amount of to get freeze-dried powder to be measured, extracts with normal butyl alcohol, filters, and filtrate is as need testing solution; The preparation of red ginseng or genseng control medicinal material solution: get red ginseng or the genseng control medicinal material is an amount of, add the methenyl choloride refluxing extraction, discard methenyl choloride liquid, residue adds water-saturated n-butanol and extracts, and extract adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medicinal material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add methyl alcohol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methanol-water 15: 40: 22: 10 was developping agent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ are dried by the fire to spot colour developing clearly, put respectively under daylight and the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 15: ginsenoside Rg in the freeze-dried powder
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatography differentiate:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, acetonitrile-water is a moving 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 chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.
Embodiment 16: the thin-layer chromatography discrimination method of the fruit of Chinese magnoliavine, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, adds ethyl acetate and extracts, and filters, and filtrate volatilizes, and residue adds the ethyl acetate dissolving, as need testing solution; The preparation of fruit of Chinese magnoliavine control medicinal material solution: it is an amount of to get fruit of Chinese magnoliavine control medicinal material, adds ethyl acetate and extracts, and filters, and filtrate volatilizes, and residue adds ethyl acetate dissolving, medicinal material solution in contrast; The preparation of reference substance solution: get schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B reference substance, add ethyl acetate respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be developping agent, launch with sherwood oil (60～90 ℃)-upper solution of 15: 5: 1 of ethyl acetate-formic acid, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 17: the liquid chromatography of schizandrin, schizandrin A, deoxyschizandrin is differentiated in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with schizandrin, schizandrin A, deoxyschizandrin reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, and acetonitrile-water is a moving phase at 60%: 40%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 18: the thin-layer chromatography discrimination method of the tuber of dwarf lilyturf in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, adds ethyl acetate and extracts, and filters, and filtrate volatilizes, and residue adds the ethyl acetate dissolving, as need testing solution; Other gets the control medicinal material tuber of dwarf lilyturf, shines medicinal material solution in pairs with legal system; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be at 85: 5: 0.3 developping agent, launch, take out, dry, put under the uviol lamp 254nm and inspect with benzene-methyl alcohol-formic acid, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color, negative noiseless;
Embodiment 19: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' the thin-layer chromatography discrimination method:
It is an amount of to get freeze-dried powder to be measured, and ethyl acetate extraction is used in water dissolving back, and extract volatilizes, and the residue dissolve with methanol is as need testing solution; Other gets ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adds methyl alcohol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel H thin layer plate, be developping agent with methenyl choloride-methanol-water at 25: 4: 1, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ dry by the fire to spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 20: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' liquid chromatography differentiate:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, and acetonitrile-1% glacial acetic acid is a moving phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 21: the thin-layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, adds 15% hydrochloric acid hydrolysis 4 hours, takes out, and puts to room temperature, regulates pH to neutral, filters, and filtrate volatilizes, and residue dissolves with methylene chloride, as need testing solution; Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adds methenyl choloride respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel H thin layer plate, be developping agent with cyclohexane-ethyl formate-water at 1: 1: 0.5, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 90 ℃ dry by the fire to spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 22: the liquid chromatography of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the round-bottomed flask, and the back that is dissolved in water is with 15% hydrochloric acid reflux 4 hours, taking-up, put to room temperature, transfer pH to neutral, extract with methylene chloride, extract volatilizes, the residue dissolve with methanol filters with miillpore filter, gets subsequent filtrate as need testing solution; Ethanolic solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, and methanol-water is a moving phase at 96: 4, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 23: red ginseng or genseng, ginsenoside Rg in the freeze-dried powder
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin-layer chromatography discrimination method:
It is an amount of to get freeze-dried powder to be measured, extracts with ethyl acetate, filters, and filtrate is as need testing solution; The preparation of red ginseng or genseng control medicinal material solution: get red ginseng or the genseng control medicinal material is an amount of, the refluxing extraction that adds methylene chloride discards dichloromethane solution, residue adds water-saturated n-butanol and extracts, and extract adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medicinal material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel H thin layer plate, with normal hexane-ethyl acetate-acetone-water 20: 30: 15: 15 was developping agent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ are dried by the fire to spot colour developing clearly, put respectively under daylight and the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 24: ginsenoside Rg in the freeze-dried powder
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatography differentiate:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, acetonitrile-0.5% glacial acetic acid aqueous solution is a moving phase, gradient elution, solvent ratios are from 0 minute to 30 minutes, and the ratio of acetonitrile is 20%, from 30 minutes to 50 minutes, the ratio of acetonitrile rises to 25% by 20%, and from 50 minutes to 70 minutes, the ratio of acetonitrile was 25%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 25%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.
Embodiment 25: the thin-layer chromatography discrimination method of the fruit of Chinese magnoliavine, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B in the parenteral solution:
It is an amount of to get parenteral solution to be measured, and the extraction that adds diethyl ether filters, and filtrate volatilizes, and residue adds the methenyl choloride dissolving, as need testing solution; The preparation of fruit of Chinese magnoliavine control medicinal material solution: it is an amount of to get fruit of Chinese magnoliavine control medicinal material, adds ethyl acetate and extracts, and filters, and filtrate volatilizes, and residue adds ethyl acetate dissolving, medicinal material solution in contrast; The preparation of reference substance solution: get schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, wuweizi ester B reference substance, add methenyl choloride respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be developping agent, launch with sherwood oil (60～90 ℃)-upper solution of 15: 5: 3 of ethyl formate-acetate, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 26: the liquid chromatography of schizandrin, schizandrin A, deoxyschizandrin is differentiated in the parenteral solution:
It is an amount of to get parenteral solution to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Ethanolic solution with schizandrin, schizandrin A, deoxyschizandrin reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, and acetonitrile-0.5% glacial acetic acid aqueous solution is a moving phase at 57%: 43%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 27: the thin-layer chromatography discrimination method of the tuber of dwarf lilyturf in the parenteral solution:
It is an amount of to get parenteral solution to be measured, and the extraction that adds methylene chloride filters, and filtrate volatilizes, and residue adds the methenyl choloride dissolving, as need testing solution; Other gets the control medicinal material tuber of dwarf lilyturf, shines medicinal material solution in pairs with legal system; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be at 70: 10: 2 developping agent, launch, take out, dry, put under the uviol lamp 254nm and inspect with methenyl choloride-ethanol-formic acid, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color, negative noiseless;
Embodiment 28: ophiopogonin B in the parenteral solution, ophiopogonin D, ophiopogonin D ' the thin-layer chromatography discrimination method:
It is an amount of to get parenteral solution to be measured, uses dichloromethane extraction, and extract volatilizes, and the residue dissolve with methanol is as need testing solution; Other gets ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adds ethanol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be at 20: 8: 2 developping agent, launch, take out with dichloromethane-ethanol-water, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ dry by the fire to spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 29: ophiopogonin B in the parenteral solution, ophiopogonin D, ophiopogonin D ' liquid chromatography differentiate:
It is an amount of to get parenteral solution to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Ethanol alcoholic solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, and acetonitrile-0.1% phosphate aqueous solution is a moving phase at 87: 13, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 30: the thin-layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the parenteral solution:
It is an amount of to get parenteral solution to be measured, adds 5% sulphuric acid hydrolysis 3 hours, takes out, and puts to room temperature, regulates pH to neutral, filters, and filtrate volatilizes, and residue dissolves with ethyl acetate, as need testing solution; Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adds ethyl acetate respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be at 2: 1: 0.2 developping agent, launch, take out with methyl alcohol-methylene chloride-water, dry, spray is with 10% sulfuric acid ethanol reagent, 90 ℃ dry by the fire to spot colour developing clear, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 31: the liquid chromatography of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in the parenteral solution:
It is an amount of to get parenteral solution to be measured, puts in the round-bottomed flask, with 5% sulphuric acid hydrolysis 3 hours, takes out, and puts to room temperature, transfers pH to neutral, extracts with ethyl acetate, and extract volatilizes, and the residue dissolve with ethanol with the miillpore filter filtration, is got subsequent filtrate as need testing solution; Ethanolic solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, and methyl alcohol-0.2% glacial acetic acid aqueous solution is a moving phase at 95: 5, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end;
Embodiment 32: red ginseng or genseng, ginsenoside Rg in the parenteral solution
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf the thin-layer chromatography discrimination method:
It is an amount of to get parenteral solution to be measured, extracts with ethyl acetate, filters, and filtrate is as need testing solution; The preparation of red ginseng or genseng control medicinal material solution: get red ginseng or the genseng control medicinal material is an amount of, the refluxing extraction that adds methylene chloride discards dichloromethane solution, residue adds ethyl acetate and extracts, and extract adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medicinal material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, with normal butyl alcohol-ethyl formate-water 20: 30: 15 upper solution be developping agent, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ are dried by the fire to spot colour developing clearly, put respectively under daylight and the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
Embodiment 33: ginsenoside Rg in the parenteral solution
1, ginsenoside Rb
1, the ginsenoside Re liquid chromatography differentiate:
It is an amount of to get parenteral solution to be measured, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, acetonitrile-0.2% phosphate aqueous solution is a moving phase, gradient elution, solvent ratios are from 0 minute to 20 minutes, and the ratio of acetonitrile is 15%, from 20 minutes to 45 minutes, the ratio of acetonitrile rises to 22% by 15%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was 22%, from 60 minutes to 80 minutes, the ratio of acetonitrile rose to 35% by 22%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatogram, tool and the reference substance chromatogram consistent chromatographic peak of time of withing a hook at the end.
Embodiment 34: ginsenoside Rg in the freeze-dried powder
1, ginsenoside Rb
1, the ginsenoside Re assay:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, acetonitrile-water is a moving 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 30 ℃; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.6mg;
(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.8mg;
(3) the per unit amount contains ginsenoside Rb
1Limit must not be less than 1.0mg;
(4) the per unit amount contains the ginsenoside Rg
1, ginsenoside Rb
1, the ginsenoside Re the limit of summation must not be less than 3.4mg.
Embodiment 35: the assay of total saponins in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the 10ml measuring bottle, adds water to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillic aldehyde-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, immediately with ice-water bath cooling 2 minutes, fixed to scale with glacial acetic acid, shake up, as need testing solution; With the ginsenoside Rg
1Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, measure absorbance log, calculate with one point external standard method at the wavelength place of 547nm, freeze-dried powder to be measured 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 20mg.
Embodiment 36: total polysaccharides assay in the freeze-dried powder:
It is an amount of that monose is got freeze-dried powder to be measured, 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg), calculate the content of monose thus;
It is an amount of that total reducing sugar is got freeze-dried powder to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 4 hours 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg, calculates sugar contents with this;
The content that deducts monose with sugar contents promptly gets the content of total polysaccharides;
Freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous dextrose, must not be less than 100mg.
Embodiment 37: total lignan assay in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, and it is fixed to scale to add water, add hydrochloric acid precipitation saponin component after, with ethyl acetate extract and and extract, water bath method, the residue dissolve with methanol is as need testing solution; With the schizandrin is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, wavelength place at 254nm measures absorbance log, calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignan in schizandrin, must not be less than 3mg.
Embodiment 38: the assay of schizandrin, schizandrin A, deoxyschizandrin in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with schizandrin, schizandrin A, deoxyschizandrin reference substance is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and methanol-water is a moving phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains schizandrin must not be less than 0.4mg;
(2) the per unit amount limit that contains schizandrin A must not be less than 0.05mg;
(3) the per unit amount limit that contains deoxyschizandrin must not be less than 0.10mg;
(4) the per unit amount limit that contains the summation of schizandrin, schizandrin A, deoxyschizandrin must not be less than 0.55mg.
Embodiment 39: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' assay:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-water is a moving phase at 90%: 10%, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains ophiopogonin B must not be less than 0.1mg;
(2) the per unit amount limit that contains ophiopogonin D must not be less than 0.1mg;
(3) the per unit amount contain ophiopogonin D ' limit must not be less than 0.02mg;
(4) the per unit amount contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.22mg.
Embodiment 40: the assay of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulfuric acid, takes out, put to room temperature, transfer pH to neutral, with methenyl choloride extract and and extract, evaporate to dryness, the residue dissolve with methanol filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-water is a moving phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method or calibration curve method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains Ruscus aculeatus L. sapogenin must not be less than 0.02mg;
(2) the per unit amount limit that contains diosgenin must not be less than 0.02mg;
(3) the per unit amount limit that contains ruscogenin must not be less than 0.02mg;
(4) the per unit amount limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.06mg.
Embodiment 41: ginsenoside Rg in the freeze-dried powder
1, ginsenoside Rb
1, the ginsenoside Re assay:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, acetonitrile-0.5% glacial acetic acid aqueous solution is a moving phase, gradient elution, solvent ratios are from 0 minute to 30 minutes, and the ratio of acetonitrile is 20%, from 30 minutes to 50 minutes, the ratio of acetonitrile rises to 25% by 20%, and from 50 minutes to 70 minutes, the ratio of acetonitrile was 25%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 25%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.6mg;
(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.8mg;
(3) the per unit amount contains ginsenoside Rb
1Limit must not be less than 1.0mg;
(4) the per unit amount contains the ginsenoside Rg
1, ginsenoside Rb
1, the ginsenoside Re the limit of summation must not be less than 3.4mg.
Embodiment 42: the assay of total saponins in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the 10ml measuring bottle, adds methyl alcohol to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillic aldehyde-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 30 minutes in 60 ℃ of water-baths, take out, immediately with ice-water bath cooling 2 minutes, fixed to scale with glacial acetic acid, shake up, as need testing solution; With ginsenoside Rb
1Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, measure absorbance log, calculate with one point external standard method at the wavelength place of 547nm, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with ginsenoside Rb
1Meter must not be less than 20mg.
Embodiment 43: total polysaccharides assay in the freeze-dried powder:
It is an amount of that monose is got freeze-dried powder to be measured, 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg), calculate the content of monose thus;
It is an amount of that total reducing sugar is got freeze-dried powder to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 2 hours 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg, calculates sugar contents with this;
The content that deducts monose with sugar contents promptly gets the content of total polysaccharides;
Freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous dextrose, must not be less than 100mg.
Embodiment 44: total lignan assay in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, and it is fixed to scale to add water, add hydrochloric acid precipitation saponin component after, with methenyl choloride extract and and extract, water bath method, the residue dissolve with methanol is as need testing solution; With the wuweizi alcohol B is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, wavelength place at 254nm measures absorbance log, calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignan in wuweizi alcohol B, must not be less than 3mg.
Embodiment 45: the assay of schizandrin, schizandrin A, deoxyschizandrin in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with schizandrin, schizandrin A, deoxyschizandrin reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, and acetonitrile-water is a moving phase at 60%: 40%, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains schizandrin must not be less than 0.4mg;
(2) the per unit amount limit that contains schizandrin A must not be less than 0.05mg;
(3) the per unit amount limit that contains deoxyschizandrin must not be less than 0.10mg;
(4) the per unit amount limit that contains the summation of schizandrin, schizandrin A, deoxyschizandrin must not be less than 0.55mg.
Embodiment 46: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' assay:
It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, and acetonitrile-1% glacial acetic acid is a moving phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains ophiopogonin B must not be less than 0.1mg;
(2) the per unit amount limit that contains ophiopogonin D must not be less than 0.1mg;
(3) the per unit amount contain ophiopogonin D ' limit must not be less than 0.02mg;
(4) the per unit amount contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.22mg.
Embodiment 47: the assay of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the freeze-dried powder:
It is an amount of to get freeze-dried powder to be measured, puts in the round-bottomed flask, and the back that is dissolved in water is with 15% hydrochloric acid reflux 4 hours, taking-up, put to room temperature, transfer pH to neutral, extract with methylene chloride, extract volatilizes, the residue dissolve with methanol filters with miillpore filter, gets subsequent filtrate as need testing solution; Ethanolic solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with eight alkyl silane bonded silica gels, and methanol-water is a moving phase at 96: 4, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method or calibration curve method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains Ruscus aculeatus L. sapogenin must not be less than 0.02mg;
(2) the per unit amount limit that contains diosgenin must not be less than 0.02mg;
(3) the per unit amount limit that contains ruscogenin must not be less than 0.02mg;
(4) the per unit amount limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.06mg.
Embodiment 48: ginsenoside Rg in the parenteral solution
1, ginsenoside Rb
1, the ginsenoside Re assay:
It is an amount of to get parenteral solution to be measured, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, acetonitrile-0.2% phosphate aqueous solution is a moving phase, gradient elution, solvent ratios are from 0 minute to 20 minutes, and the ratio of acetonitrile is 15%, from 20 minutes to 45 minutes, the ratio of acetonitrile rises to 22% by 15%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was 22%, from 60 minutes to 80 minutes, the ratio of acetonitrile rose to 35% by 22%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; Calculate with one point external standard method, parenteral solution to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.6mg;
(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.8mg;
(3) the per unit amount contains ginsenoside Rb
1Limit must not be less than 1.0mg;
(4) the per unit amount contains the ginsenoside Rg
1, ginsenoside Rb
1, the ginsenoside Re the limit of summation must not be less than 3.4mg.
Embodiment 49: the assay of total saponins in the parenteral solution:
It is an amount of to get parenteral solution to be measured, puts in the 10ml measuring bottle, adds ethanol to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 10% vanillic aldehyde-glacial acetic acid solution 0.3ml, perchloric acid 3.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, immediately with the ice-water bath cooling, fixed to scale with glacial acetic acid, shake up, as need testing solution; With ginsenoside Re is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, wavelength place at 547nm measures absorbance log, calculate with one point external standard method, parenteral solution to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total saponins in the ginsenoside Re, must not be less than 20mg.
Embodiment 50: total polysaccharides assay in the parenteral solution:
It is an amount of that monose is got parenteral solution to be measured, puts in the iodine flask, and 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, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate vs immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank test, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg), calculate the content of monose thus;
It is an amount of that total reducing sugar is got parenteral solution to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 3 hours 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 vs 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 test, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous dextrose of 9.008mg, calculates sugar contents with this;
The content that deducts monose with sugar contents promptly gets the content of total polysaccharides;
Parenteral solution to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous dextrose, must not be less than 100mg.
Embodiment 51: total lignan assay in the parenteral solution:
It is an amount of to get parenteral solution to be measured, puts in the measuring bottle, and it is fixed to scale to add water, add hydrochloric acid precipitation saponin component after, with methylene chloride extract and and extract, water bath method, the residue dissolve with methanol is as need testing solution; With the schizandrin is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, wavelength place at 254nm measures absorbance log, calculate with one point external standard method, parenteral solution to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignan in schizandrin, must not be less than 3mg.
Embodiment 52: the assay of schizandrin, schizandrin A, deoxyschizandrin in the parenteral solution:
It is an amount of to get parenteral solution to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Ethanolic solution with schizandrin, schizandrin A, deoxyschizandrin reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, and acetonitrile-0.5% glacial acetic acid aqueous solution is a moving phase at 57%: 43%, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, parenteral solution to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains schizandrin must not be less than 0.4mg;
(2) the per unit amount limit that contains schizandrin A must not be less than 0.05mg;
(3) the per unit amount limit that contains deoxyschizandrin must not be less than 0.10mg;
(4) the per unit amount limit that contains the summation of schizandrin, schizandrin A, deoxyschizandrin must not be less than 0.55mg.
Embodiment 53: ophiopogonin B in the parenteral solution, ophiopogonin D, ophiopogonin D ' assay:
It is an amount of to get parenteral solution to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; Ethanol alcoholic solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, and acetonitrile-0.1% phosphate aqueous solution is a moving phase at 87: 13, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, parenteral solution to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains ophiopogonin B must not be less than 0.1mg;
(2) the per unit amount limit that contains ophiopogonin D must not be less than 0.1mg;
(3) the per unit amount contain ophiopogonin D ' limit must not be less than 0.02mg;
(4) the per unit amount contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.22mg.
Embodiment 54: the assay of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the parenteral solution:
It is an amount of to get parenteral solution to be measured, puts in the round-bottomed flask, with 5% sulphuric acid hydrolysis 3 hours, takes out, and puts to room temperature, transfers pH to neutral, extracts with ethyl acetate, and extract volatilizes, and the residue dissolve with ethanol with the miillpore filter filtration, is got subsequent filtrate as need testing solution; Ethanolic solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with the dialkyl silane bonded silica gel, and methyl alcohol-0.2% glacial acetic acid aqueous solution is a moving phase at 95: 5, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, parenteral solution to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:
(1) the per unit amount limit that contains Ruscus aculeatus L. sapogenin must not be less than 0.02mg;
(2) the per unit amount limit that contains diosgenin must not be less than 0.02mg;
(3) the per unit amount limit that contains ruscogenin must not be less than 0.02mg;
(4) the per unit amount limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.06mg.
Claims (4)
1. the detection method of a pulse restoring injection comprises finger-print test, differential test method and content test method, it is characterized in that:
A, adopt liquid phase chromatography test red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics be main finger-print:
(1) preparation of need testing solution: it is an amount of to get Shengmai injection to be measured, adds water or methyl alcohol or 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 reference substance in an amount of red ginseng or genseng and the tuber of dwarf lilyturf medicinal material, comprise the reference substance ginsenoside Rg
1, ginsenoside Rb
1, a kind of among the ginsenoside Rf, water or methyl alcohol or dissolve with ethanol are settled to suitable concn, as object of reference solution;
(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase A is 0.01mol/L～2mol/L biphosphate sodium water solution or 0.01mol/L～2mol/L potassium dihydrogen phosphate aqueous solution or 0.01mol/L～2mol/L sodium hydrogen phosphate aqueous solution or water or 0.1%～5% glacial acetic acid solution or 0.1%～5% formic acid solution or 0.02%～5% phosphoric acid solution; B is 10%～100% acetonitrile solution or 10%～absolute methanol solution, and gradient elution, flow velocity be 0.5～2.0ml/min, detect wavelength is that one or several or evaporation photodetector in 190～410nm scope detects, and column temperature is in 20～60 ℃ of scopes;
(4) formulation of standard finger-print: with said method as formulate based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～50;
(5) based on the described method in (1)～(3) as red ginseng or genseng in the injection to be measured and the tuber of dwarf lilyturf composition characteristics the means of testing of finger-print, the finger-print of preparation testing sample;
(6) with the finger-print and the contrast of above-mentioned standard finger-print of injection to be measured, should meet the following requirements:
I. calculate the similarity of the finger-print and the standard finger-print of injection to be measured, should be 0.80～1.00;
II. in the injection finger-print to be measured, non-total peak area must not surpass 10% of total peak area;
In the ratio that 30%～80% total peak area arranged in the injection finger-print III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than soil 50%;
The discrimination method of B, described injection comprises following content:
The thin-layer chromatography discrimination method of the fruit of Chinese magnoliavine in a, the injection:
It is an amount of to get Shengmai injection to be measured, adds methenyl choloride or ether or ethyl acetate or normal hexane or 10%～absolute ethyl alcohol or 10%～absolute methanol and extracts, and filters, and filtrate volatilizes, and residue adds methenyl choloride or ethyl acetate dissolving, as need testing solution; Other gets one or more preparation contrast solutions in fruit of Chinese magnoliavine control medicinal material, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B; The preparation of fruit of Chinese magnoliavine control medicinal material solution: it is an amount of to get fruit of Chinese magnoliavine control medicinal material, add methenyl choloride or ether or ethyl acetate or normal hexane or 10%～absolute ethyl alcohol or 10%～absolute methanol and extract, filter, filtrate volatilizes, residue adds methenyl choloride or ethyl acetate dissolving, medicinal material solution in contrast; The preparation of reference substance solution: get one or more reference substances in schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B; Add methenyl choloride or ethyl acetate respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned need testing solution and reference substance solution, put respectively in same silica gel g thin-layer plate or silica gel H thin layer plate or silica G F
254On the thin layer plate, sherwood oil-ethyl formate or ethyl acetate-formic acid or acetate 2～40: 0.2～15 with 30～60 ℃ sherwood oils or 60～90 ℃: 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 acetate 0.2～5: 0.5～10: 1～15: 0.1～5 are developping agent, launch, take out, dry, putting uviol lamp 365nm or 254nm inspects or sprays with 2%～20% chromotropic acid-concentrated sulfuric acid solution or 2%～20% phosphomolybdic acid ethanol solution or anisaldehyde sulfuric acid solution, dry by the fire the clear or smoked colour developing of iodine at 80 ℃～160 ℃ to the spot colour developing, in the test sample chromatogram, with control medicinal material chromatogram or the corresponding position of reference substance chromatogram on, should show the spot of same color;
The thin-layer chromatography discrimination method of the tuber of dwarf lilyturf in b, the injection:
It is an amount of to get Shengmai injection to be measured, adds methenyl choloride-methyl alcohol mixed solution or normal butyl alcohol or methenyl choloride or ethyl acetate or methylene chloride and extracts, as need testing solution; Other gets the control medicinal material tuber of dwarf lilyturf, adds methenyl choloride-methyl alcohol mixed solution or ethyl acetate or normal butyl alcohol or methenyl choloride or methylene chloride and extracts, and filters, and evaporate to dryness, residue add methenyl choloride or methylene chloride dissolving, medicinal material solution in contrast; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H thin layer plate or silica G F
254On the thin layer plate, with benzene or toluene or methenyl choloride or methylene chloride-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 developping agent, launch, take out, dry, put and inspect under uviol lamp 365nm or the 254nm or spray with 1%～50% sulfuric acid or vanillic aldehyde reagent or 1%～25% vanillin reagent or anisaldehyde reagent, it is clear to dry by the fire to the spot colour developing at 80 ℃～160 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, should show the fluorescence spot of same color;
The thin-layer chromatography discrimination method of genseng or red ginseng in c, the injection:
It is an amount of to get Shengmai injection to be measured, with normal butyl alcohol or ethanol or methyl alcohol or ethyl acetate or methenyl choloride or methylene chloride extraction, filters, and filtrate is as need testing solution; Other gets red ginseng or genseng control medicinal material, ginsenoside Rg
1, ginsenoside Rb
1, among the ginsenoside Re, ginsenoside Rf one or more, the preparation contrast solution; The preparation of red ginseng or genseng control medicinal material solution: get red ginseng or the genseng control medicinal material is an amount of, adding methenyl choloride or methylene chloride reflux extracts, discard methenyl choloride or dichloromethane solution, residue adds water-saturated n-butanol or ethyl acetate extracts, extract adds ammonia solution, divides and gets upper strata, evaporate to dryness, residue is with methyl alcohol or dissolve with ethanol, medicinal material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg
1, ginsenoside Rb
1, one or more reference substance among the ginsenoside Re, ginsenoside Rf, add methyl alcohol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H thin layer plate or silica gel 6F
254On the thin layer plate, lower floor's solution or normal 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 methenyl choloride or methylene chloride or normal hexane or cyclohexane-ethyl acetate or ethyl formate-methyl alcohol or ethanol or acetone-water: 1～15 upper strata is a developping agent, launch, take out, dry, spray is with 5%～50% sulfuric acid ethanol reagent, it is clear to dry by the fire to the spot colour developing at 80 ℃～160 ℃, put respectively under daylight and the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, should show the spot of same color;
C, described injection content detecting method should comprise:
Ginsenoside Rg in a, the injection
1, ginsenoside Re, ginsenoside Rb
1Assay:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methyl alcohol or moving phase or ethanol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Re, ginsenoside Rb
1In the methyl alcohol of one or more reference substances or ethanolic solution be contrast, adopt liquid phase chromatography, chromatographic column is a filling agent with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methyl alcohol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% formic acid solution 5%～40%: 95%～60% are moving phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with external standard method or calibration curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be:
(1) the per unit amount contains the ginsenoside Rg
1Limit must not be less than 0.8mg;
(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.4mg;
(3) the per unit amount contains ginsenoside Rb
1Limit must not be less than 0.5mg;
(4) the per unit amount contains the ginsenoside Rg
1, the ginsenoside Re the limit of summation must not be less than 1.7mg;
The assay of total saponins in b, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, and adding distil water or methyl alcohol or ethanol make dissolving and fixed to scale in right amount, shake up, precision is measured in right amount, puts in the measuring bottle, water bath method takes out immediately, and precision adds 1%～50% vanillic aldehyde-glacial acetic acid solution 0.1ml～10ml, perchloric acid 0.1ml～15ml shakes up, and heats 3～50 minutes in 30 ℃～80 ℃ water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with the ginsenoside Rg
1Or ginsenoside Rb
1Or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures absorbance log, calculate with external standard method or calibration curve method, Shengmai injection to be measured 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 10mg;
Total polysaccharides assay in c, the injection:
It is an amount of that monose is got Shengmai injection to be measured, puts in the iodine flask, and pH is regulated to neutral with sodium hydroxide test solution in adding distil water dissolving back, the accurate iodine liquid 25ml that adds 0.1mol/L shakes up, and dropwise adds 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 vs immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank test, promptly to blue; The iodine liquid of every 1ml0.1mol/L is equivalent to the anhydrous dextrose of 9.008mg, calculates the content of monose thus;
It is an amount of that total reducing sugar is got Shengmai injection to be measured, put in the iodine flask, adding distil water adds dilute sulfuric acid 25ml after making dissolving, reflux 1～4 hour, put cold, add 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, and precision adds 0.1mol/L iodine liquid 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 vs immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank test, promptly to blue; The iodine liquid of every 1ml 0.1mol/L is equivalent to the anhydrous dextrose of 9.008mg, calculates sugar contents with this;
The content that deducts monose with sugar contents promptly gets the content of total polysaccharides;
Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous dextrose, must not be less than 50mg;
The all or part of total content of surveying composition of the saponins in the described injection, polysaccharide accounts for more than 25% of total solid of deducting auxiliary material amount and amount of moisture in the preparation.
2. according to the detection method of the described pulse restoring injection of claim 1, it is characterized in that: adopt liquid chromatography for measuring based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics finger-print:
(1) preparation of need testing solution: it is an amount of that precision takes by weighing Shengmai injection to be measured, adds water and make the solution that every 1ml contains 50mg, with the miillpore filter 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 methyl alcohol 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 is 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) formulation of standard finger-print: with said method as formulate based on red ginseng or genseng and the tuber of dwarf lilyturf composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;
(5) based on the described method in (1)～(3) as red ginseng or genseng in the injection to be measured and the tuber of dwarf lilyturf composition characteristics the means of testing of finger-print, the finger-print of preparation testing sample;
(6) with pulse restoring injection finger-print to be measured and the contrast of above-mentioned standard finger-print, should meet the following requirements:
I. calculate the similarity of the finger-print and the standard finger-print of injection to be measured, should be 0.90～1.00;
II. in the injection finger-print to be measured, non-total peak area must not surpass 5% of total peak area;
In the ratio that 40%～60% total peak area arranged in the injection finger-print III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than soil 30%.
3. according to the detection method of the described pulse restoring injection of claim 1, it is characterized in that: the discrimination method of described injection:
The thin-layer chromatography discrimination method of the fruit of Chinese magnoliavine in a, the injection:
It is an amount of to get Shengmai injection to be measured, adds methenyl choloride and extracts, and filters, and filtrate volatilizes, and residue adds the methenyl choloride dissolving, as need testing solution; Other gets one or more preparation contrast solutions in fruit of Chinese magnoliavine control medicinal material, schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B, the preparation of fruit of Chinese magnoliavine control medicinal material solution: it is an amount of to get fruit of Chinese magnoliavine control medicinal material, adding methenyl choloride extracts, filter, filtrate volatilizes, residue adds methenyl choloride dissolving, medicinal material solution in contrast; The preparation of reference substance solution: get one or more reference substances in schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C, Schisantherin C, the wuweizi ester B, add methenyl choloride respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be developping agent, launch with sherwood oil-ethyl formates of 30～60 ℃-upper solution of 15: 5: 1 of formic acid, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color;
The thin-layer chromatography discrimination method of the tuber of dwarf lilyturf in b, the injection:
It is an amount of to get Shengmai injection to be measured, adds 7: 3 mixed solutions of methenyl choloride-methyl alcohol and extracts, and filters, and filtrate volatilizes, and residue adds the methenyl choloride dissolving, as need testing solution; Other gets the control medicinal material tuber of dwarf lilyturf, shines medicinal material solution in pairs with legal system; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica G F respectively
254On the thin layer plate, be at 80: 5: 0.1 developping agent, launch, take out, dry, put under the uviol lamp 254nm and inspect with toluene-methyl alcohol-glacial acetic acid, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color, negative noiseless;
The thin-layer chromatography discrimination method of genseng or red ginseng in c, the injection:
It is an amount of to get Shengmai injection to be measured, extracts with normal butyl alcohol, filters, and filtrate is as need testing solution; Preparation control medicinal material solution; The preparation of red ginseng or genseng control medicinal material solution: get red ginseng or the genseng control medicinal material is an amount of, add the methenyl choloride refluxing extraction, discard methenyl choloride liquid, residue adds water-saturated n-butanol and extracts, and extract adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medicinal material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg
1, ginsenoside Rb
1, ginsenoside Re, ginsenoside Rf's reference substance, add methyl alcohol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin-layered chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride-ethyl acetate-methanol-water 15: 40: 22: 10 was developping agent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulfuric acid ethanol reagent, 105 ℃ are dried by the fire to spot colour developing clearly, put respectively under daylight and the ultraviolet lamp 365nm and inspect, in the test sample chromatogram, with control medicinal material chromatogram and the corresponding position of reference substance chromatogram on, show the spot of same color.
4. according to the detection method of the described pulse restoring injection of claim 1, it is characterized in that: the method for testing of described injection content should comprise:
Ginsenoside Rg in a, the injection
1, ginsenoside Re, ginsenoside Rb
1Assay:
It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methyl alcohol to scale, shakes up, and filters with miillpore filter, gets subsequent filtrate as need testing solution; With the ginsenoside Rg
1, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid phase chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filling agent, acetonitrile-water is a moving 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 30 ℃; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be:
(1) the per unit amount contains the ginsenoside Rg
1Limit must not be less than 1.6mg;
(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.8mg;
(3) the per unit amount contains ginsenoside Rb
1Limit must not be less than 1.0mg;
(4) the per unit amount contains the ginsenoside Rg
1, ginsenoside Rb
1, the ginsenoside Re the limit of summation must not be less than 3.4mg;
The assay of total saponins in b, the injection:
It is an amount of to get Shengmai injection to be measured, puts in the 10ml measuring bottle, adds water to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillic aldehyde-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, immediately with ice-water bath cooling 2 minutes, fixed to scale with glacial acetic acid, shake up, as need testing solution; With the ginsenoside Rg
1Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotometric method of Chinese Pharmacopoeia appendix, measure absorbance log, calculate with one point external standard method at the wavelength place of 547nm, Shengmai injection to be measured 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 20mg;
Total polysaccharides assay in c, the injection:
It is an amount of that monose is got Shengmai injection to be measured, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and the accurate iodine liquid 25ml that adds 0.1mol/L 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 vs 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 test, promptly; Every 1ml0.1mol/L iodine liquid be equivalent to the anhydrous dextrose of 9.008mg, calculate the content of monose thus;
It is an amount of that total reducing sugar is got Shengmai injection to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 4 hours is put coldly, adds 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, the accurate iodine liquid 25ml that adds 0.1mol/L 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 vs 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 test, promptly; The iodine liquid of every 1ml 0.1mol/L is equivalent to the anhydrous dextrose of 9.008mg, calculates sugar contents with this;
The content that deducts monose with sugar contents promptly gets the content of total polysaccharides;
Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous dextrose, must not be less than 100mg;
The all or part of total content of surveying composition of the saponins in the described injection, polysaccharide accounts for more than 25% of total solid of deducting auxiliary material amount and amount of moisture in the preparation.
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| CNB2005102007595A CN100529753C (en) | 2004-12-13 | 2005-12-02 | Quality controlling method for pulse restoring injection |
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| CN1970035B (en) * | 2006-12-19 | 2010-09-08 | 贵州益佰制药股份有限公司 | Formulation of 'Sheng Mai', preparation process and quality control method thereof |
| CN101101280B (en) * | 2007-07-10 | 2010-07-28 | 正大青春宝药业有限公司 | Use of pulse beat restoring capsule fingerprint pattern detect technology |
| CN101879270B (en) * | 2009-05-07 | 2014-02-12 | 天津天士力之骄药业有限公司 | Traditional Chinese medicine injectable powder and quality control method thereof |
| CN102706998A (en) * | 2012-02-15 | 2012-10-03 | 江苏苏中药业集团股份有限公司 | Quality control method for shengmai injection |
| WO2015042852A1 (en) * | 2013-09-27 | 2015-04-02 | 四川川大华西药业股份有限公司 | Method for detecting pulse-activating injection intermediates |
| CN104407088A (en) * | 2014-12-04 | 2015-03-11 | 天津大学 | Quantitative analysis method for dioscin in compounded traditional Chinese medicine preparation |
| 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 |
| CN109541098B (en) * | 2018-11-27 | 2020-11-10 | 华润三九（雅安）药业有限公司 | Radix ophiopogonis fingerprint spectrum, construction method thereof and radix ophiopogonis quality detection method |
| CN109541117B (en) * | 2018-12-21 | 2021-01-29 | 青海普兰特药业有限公司 | Detection method of lung-moistening pharmaceutical composition |
| 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 |
| CN110749693A (en) * | 2019-10-23 | 2020-02-04 | 葵花药业集团（襄阳）隆中有限公司 | Thin-layer chromatography identification method for radix ophiopogonis component in Erdong decoction formula |
| CN112326861A (en) * | 2020-11-26 | 2021-02-05 | 太极集团·四川天诚制药有限公司 | Quality standard detection method of pulse-activating decoction |
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