CN107998177A - Children open larynx sword spray and its prepare and the quality standard method of inspection - Google Patents
Children open larynx sword spray and its prepare and the quality standard method of inspection Download PDFInfo
- Publication number
- CN107998177A CN107998177A CN201711436355.5A CN201711436355A CN107998177A CN 107998177 A CN107998177 A CN 107998177A CN 201711436355 A CN201711436355 A CN 201711436355A CN 107998177 A CN107998177 A CN 107998177A
- Authority
- CN
- China
- Prior art keywords
- solution
- reference substance
- methanol
- test
- matrine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/534—Mentha (mint)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/489—Sophora, e.g. necklacepod or mamani
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/12—Aerosols; Foams
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Insects & Arthropods (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a kind of children to open larynx sword spray and its preparation and the quality standard method of inspection, it is by Crisped Ardisia Ardisia gigantifolia, subprostrate sophora, cicada slough and four taste Chinese medicine of menthol composition, in discrimination process, the thin layer for having revised bicolor differentiates, differentiated in primary standard by control of Bergenin, test sample preparation method is complicated, and now former method is revised, adds bicolor control medicinal material, simplify test sample preparation method, this method specificity is strong, favorable reproducibility, therefore is included in standard body；Language description, which has carried out specification, to be differentiated to the thin layer of menthol, and solvent agents useful for same benzene is changed to toluene；The thin layer for adding subprostrate sophora differentiates, using matrine as control, stablizes through investigating this method, favorable reproducibility, therefore be included in standard body.Separately the thin layer discriminating to cicada slough is studied.
Description
Technical field
The present invention relates to medicine prepare and its Quality Control Technology field, especially a kind of children open larynx sword spray and its
Prepare and the quality standard method of inspection.
Background technology
Open larynx sword spray（Child form）Quality standard is now recorded in State Food and Drug Administration's national drug mark
Quasi- WS-10132 (ZD-0132) -2002-2012Z, the approval number of the drug are Chinese medicines quasi-word Z20025142；This product is by four taste Chinese medicines
Form, differentiated in primary standard by control of Bergenin, test sample preparation method is complicated, and is short of cinnabar with control medicinal material
Discrimination method, while it is short of the content assaying method to bicolor active ingredient Bergenin；Menthol is adopted in proper mass standard
Solvent is reagent benzene, while is short of and the thin layer of subprostrate sophora is differentiated.
The content of the invention
The technical problems to be solved by the invention are：A kind of children are provided and open larynx sword spray and its preparation and quality standard
The method of inspection, with overcome the deficiencies in the prior art.
What the present invention was realized in：
A kind of children open larynx sword spray, calculate by weight, it includes 200-300 parts of Crisped Ardisia Ardisia gigantifolia, 200-300 parts of subprostrate sophora,
0.5-2 parts of 150-250 parts of cicada slough and menthol.
A kind of children open the preparation method of larynx sword spray, in addition to menthol, by three taste of Crisped Ardisia Ardisia gigantifolia, subprostrate sophora and cicada slough
Add water to cook it is secondary, for the first time 2 it is small when, second 1 it is small when, collecting decoction, filtration, filtrate be concentrated into relative density for 1.05~
1.10（50℃）Clear cream, add ethanol, make alcohol content up to 80%, stand 24 it is small when, filtration, decompression filtrate recycling ethanol is simultaneously dense
Relative density is reduced to as 1.10~1.20（80℃）Clear cream, take menthol, sodium benzoate 0.5-1.5g, citric acid 0.5-
1.5g, flavoring pineapple essence 5-6ml, with ethanol 20ml, after stirring and dissolving, add in above-mentioned medicinal extract, add water to 1000ml, stirring is equal
Even, filtration is filling, to obtain the final product.
A kind of children open the quality standard method of inspection of larynx sword spray, which opens larynx sword spray by Crisped Ardisia Ardisia gigantifolia,
Subprostrate sophora, cicada slough and four taste Chinese medicine of menthol composition, differentiate specific as follows：
1）, bicolor thin layer differentiate：Take this product2ml, it is evaporated, residue adds methanol1mlMake dissolving, as test solution；Separately
TakeBicolor control medicinal material 0.5g, adds methanol 20ml, is ultrasonically treated 30 minutes, lets cool, and filters, and filtrate is evaporated, and residue adds methanol 1ml makes dissolving, as control medicinal material solution；Take againBergenin reference substance, adds methanol that solution of every 1ml containing 0.5mg is made；According to
Thin-layered chromatography is tested, and draws above-mentioned each 3 μ l of three kinds of solution, is put respectively on same silica gel g thin-layer plate, with chloroform-second
Acetoacetic ester-methanol (5:4:2) it is solvent, is unfolded, takes out, dry, spray with -1% potassium ferricyanide solution of 1% liquor ferri trichloridi
(1:1) mixed solution；In test sample chromatography, on position corresponding with control medicinal material and reference substance chromatography, same color is shown
Spot；
2）The thin layer of menthol differentiates：This product 10ml is taken, with ether shaking extraction 2 times, each 20ml, is volatilized naturally, residue adds
Methanol 2ml makes dissolving, as test solution；Menthol reference substance separately is taken, adds methanol that solution of every 1ml containing 2mg is made, as
Reference substance solution；Tested according to thin-layered chromatography, draw 10 μ l of test solution, 5 μ l of reference substance solution, put respectively in same silica gel
On G lamellaes, with toluene-ethyl acetate（19:1）For solvent, it is unfolded, takes out, dry, spray with vanillin-sulfuric acid test solution-second
Alcohol（1:4）, clear spot is heated at 100 DEG C；In test sample chromatography, on position corresponding with reference substance chromatography, show identical
The spot of color；
3）The thin layer of subprostrate sophora differentiates：Matrine and test solution 5ml under oxymatrine item are taken, is evaporated, residue adds trichlorine
Methane 1ml makes dissolving, as test solution；Matrine reference substance separately is taken, adds chloroform that solution of every 1ml containing 1mg is made,
As reference substance solution；Tested according to thin-layered chromatography, draw 3 μ l of test solution, 2 μ l of reference substance solution, put respectively in same
On silica gel g thin-layer plate, with chloroform-methanol-strong ammonia solution（12:1:0.1）For solvent, be unfolded, take out, dry, spray with
Dilute bismuth potassium iodide test solution；In test sample chromatography, with reference substance chromatography relevant position, showing identical orange-yellow spot；
4）The discriminating of cicada slough
Mainly include amino acid in cicada slough, using liquid phase mirror method for distinguishing, 17 kinds of amino acid are screened, method is as follows：
Chromatographic condition chromatographic column：113#Kromasil100-5C18 (4.6 × 250mm, 5 μm) mobile phase：30mM sodium acetates（36%
Acetic acid tune pH to 6.50）：Acetonitrile 355：14 be mobile phase A, and 80% acetonitrile is Mobile phase B, carries out gradient elution：40 DEG C of column temperature；Stream
Fast 1.0ml/min；5 μ l of sample size；Detection wavelength 254nm；
Deriving method：Accurate 100 μ l of mixed reference substance solution, the 400 μ l of test solution of measuring adds in 10ml centrifuge tubes respectively
Enter 0.1mol/LHCl and supply 1ml, vortex mixed uniformly adds tri- second of 0.1mol/LPITC acetonitrile solutions 0.5ml, 1.0mol/L afterwards
Amine acetonitrile solution 0.5ml, vortex mixed is uniform, room temperature place 1 it is small when after, add 4ml n-hexanes, vortex mixed is uniform, room temperature
Place after ten minutes, draw lower floor's solution, to obtain the final product.
Foregoing children are opened in the quality standard method of inspection of larynx sword spray, the content assaying method of the Bergenin
It is：According to according to liquid chromatography for measuring, specifically,
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；With methanol-water（23:77）For
Mobile phase；Detection wavelength is 275nm.Number of theoretical plate is calculated by Bergenin peak should be not less than 1500；
The preparation of reference substance solution takes Bergenin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made and contains Bergenin 80
The solution of μ g, to obtain the final product；
The preparation of test solution takes this product under content uniformity item, shakes up, and precision measures this product 2ml, puts in 10ml measuring bottles, adds
Methanol shakes up to scale, filters, take subsequent filtrate, to obtain the final product；
Determination method difference is accurate to draw reference substance solution and each 5 μ l of test solution, injects liquid chromatograph, measures, to obtain the final product；
This product contains bicolor per 1ml in terms of Bergenin (C14H16O9), must not be less than 0.25mg.
Foregoing children are opened in the quality standard method of inspection of larynx sword spray, and the rock matrine and oxymatrine contain
Quantity measuring method is：According to high effective liquid chromatography for measuring, specifically,
Chromatographic condition and system suitability are using amino bonded silica gel as filler, with acetonitrile-phosphoric acid solution of isopropanol -3%
(85:5:10) it is mobile phase；Detection wavelength is 210nm.Number of theoretical plate is calculated by matrine peak should be not less than 4000；
The preparation of reference substance solution takes matrine, oxymatrine reference substance appropriate, accurately weighed, adds acetonitrile-absolute ethyl alcohol
（80:20）It is respectively prepared every 1ml 0.1mg containing matrine, the mixed solution of oxymatrine 0.075mg, to obtain the final product.
The preparation of test solution takes this product under content uniformity item, shakes, and precision measures 2ml, adds 2g neutral aluminas
Aluminium（100-200 mesh）Mix thoroughly, put 70 DEG C of water bath methods, be added in neutral alumina column（100-200 mesh, 2g, internal diameter 1.5cm）On,
Successively with chloroform, chloroform:Methanol（7:3）Each 20ml elutions, collect eluent, put 70 DEG C of water bath methods, residue adds
Acetonitrile-absolute ethyl alcohol（80:20）Dissolving, is transferred in 10ml measuring bottles, adds acetonitrile-absolute ethyl alcohol（80:20）To scale, shake,
Filtration, takes subsequent filtrate, to obtain the final product；
Determination method is accurate respectively to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures, calculates,
To obtain the final product；
This product contains subprostrate sophora per 1ml in terms of the total amount of matrine (C15H24N2O) and oxymatrine (C15H24N2O2), must not
Less than 0.15mg.
By adopting the above-described technical solution, compared with prior art, the thin layer that the present invention has revised bicolor differentiates, former
Differentiated in standard by control of Bergenin, test sample preparation method is complicated, and now former method is revised, is added
Bicolor control medicinal material, simplifies test sample preparation method, this method specificity is strong, favorable reproducibility, therefore is included in standard body；It is right
The thin layer of menthol differentiates that language description has carried out specification, and solvent agents useful for same benzene is changed to toluene；Add mountain beans
The thin layer of root differentiates, using matrine as control, stablizes through investigating this method, favorable reproducibility, therefore be included in standard body.
Brief description of the drawings
Attached drawing 1 is 17 kinds of amino acid and to open larynx sword sample in the present invention（Lot number：20160202）Chromatogram compare；
Attached drawing 2 is that the discrimination method of cicada slough in the present invention gropes 1；
Attached drawing 3 is that the discrimination method of cicada slough in the present invention gropes 2；
Attached drawing 4 is that the discrimination method of cicada slough in the present invention gropes 3；
Attached drawing 5 is Bergenin uv scan figure in the present invention;
Attached drawing 6 is that specificity investigates reference substance, test sample, negative sample HPLC comparison diagrams in the present invention；
Attached drawing 7 is Bergenin Peak homogeneity in the present invention（Test sample）；
Attached drawing 8 is Bergenin standard curve in the present invention；
Attached drawing 9 is that durability investigates different chromatographic column HPLC contrasts chromatograms in the present invention；
Attached drawing 10 is that durability investigates different column temperature HPLC contrasts chromatograms in the present invention；
Attached drawing 11 is that durability investigates HPLC contrasts chromatogram different in flow rate in the present invention
Attached drawing 12 is that durability investigates different mobile phase ratio HPLC contrasts chromatograms in the present invention；
Attached drawing 13 is matrine in the present invention, oxymatrine uv scan figure；
Attached drawing 14 is that specificity investigates reference substance, test sample, negative sample HPLC comparison diagrams in the present invention；
Attached drawing 15 is matrine Peak homogeneity in the present invention（Test sample）；
Attached drawing 16 is matrine standard curve in the present invention；
Attached drawing 17 is oxymatrine standard curve in the present invention；
Attached drawing 18 is that durability investigates different chromatographic column HPLC contrasts chromatograms in the present invention；
Attached drawing 19 is that durability investigates different mobile phase ratio HPLC contrasts chromatograms in the present invention；
Attached drawing 20 is that durability investigates HPLC contrasts chromatogram different in flow rate in the present invention.
Embodiment
The embodiment of the present invention：
Open larynx sword spray（Child form）Middle correlation Chinese medicine introduction
1.1 bicolor main chemical compositions and physicochemical property
Bicolor is the dry root of Myrsinacea plant bicolor Ardisia crenata Sims.
Chemical composition：Triterpene saponin, Coumarins and other classes are mainly contained in bicolor；Wherein saponin(e structure type
Predominantly pentacyclic triterpene oleanane type derivative, its aglycon have 2 types：Ether epoxy and 12- alkene.Coumarins is mainly
Bergenin.Also contain some other constituents：Japanese ardisia quinone, freidelin, cupreol, daucusin,.
Bergenin
【Structural formula】
【Molecular formula and molecular weight】C14H16O9；328.27
【Physical behavior】The acicular crystal or crystalline powder of white loose, 232~240 DEG C of fusing point, are dissolved in methanol, are slightly soluble in water
And ethanol.
1.2 subprostrate sophora main chemical compositions and physicochemical property
Subprostrate sophora is the dry root and rhizome of legume sophora tonkinensis Gapnep Sophora tonkinensis Gagnep..
Chemical composition：Contain the chemical compositions [1] such as the compounds such as alkaloid, saponin(e, flavonoids and polysaccharide in subprostrate sophora, it
Main medicinal component be matrine in alkaloid, based on oxymatrine.
Matrine
【Structural formula】
【Molecular formula and molecular weight】C15H24N2O；248.37
【Physical behavior】White powder, 84 DEG C of fusing point, can be dissolved in water, benzene, chloroform, methanol, ethanol, be slightly soluble in petroleum ether.
Oxymatrine
【Structural formula】
【Molecular formula and molecular weight】C15H24N2O2；264.36
【Physical behavior】Colourless column crystallization, 162 DEG C~163 DEG C of fusing point（Hydrate）, 207 DEG C（Anhydride）It is dissolved in water, chloroform,
Ethanol, is insoluble in ether, methyl ether, petroleum ether.
1.3 research conditions related with quality standard revision
1.3.1 thin layer differentiates item
（1）Bicolor thin-layer identification method is revised：Bicolor control medicinal material is added, and simplifies discrimination method.
（2）Specification menthol thin layer differentiates language description：" reference substance solution that 2mg/ml is made " is changed to " add methanol
Solution of every 1ml containing 2mg is made, as reference substance solution "；And solvent is revised：By " benzene-ethyl acetate（19:1）" be changed to
" toluene-ethyl acetate（19:1）”.
（3）The thin layer for adding subprostrate sophora in finished product differentiates.
（4）The discriminating of cicada slough thin layer is carried out：Bibliography and research process etc. is described in detail.
1.3.2 assay item
（1）Determination of Bergenin measure is added in preparation, methodology validation experiment is completed, by measuring rock in 15 batches of finished products
Chinese cabbage cellulose content, fixes tentatively this product and contains bicolor per 1ml with Bergenin（C14H16O9）Meter, must not be less than 0.25mg.
（2）The content assaying method of subprostrate sophora index components in preparation is revised：Because primary standard method can only extract hardship
Join alkali, in order to ensure the accuracy and integrality of sample measure, its method is revised, method can ensure in preparation after revision
Matrine and oxymatrine extraction are complete, and limit is with matrine and oxymatrine concentration summation meter, and by 15 batches
The content of matrine and oxymatrine in finished product, fixes tentatively this product and contains subprostrate sophora per 1ml with matrine (C15H24N2O) and oxidation
The total amount meter of matrine (C15H24N2O2).
Open larynx sword spray（Child form）And its preparation process
Open larynx sword spray（Child form）Calculate by weight, it includes 200-300 parts of Crisped Ardisia Ardisia gigantifolia, 200-300 parts of subprostrate sophora, cicada
Slough off 150-250 parts and 0.5-2 parts of menthol.
Open larynx sword spray（Child form）Preparation method, in addition to menthol, by three taste of Crisped Ardisia Ardisia gigantifolia, subprostrate sophora and cicada slough
Add water to cook it is secondary, for the first time 2 it is small when, second 1 it is small when, collecting decoction, filtration, filtrate be concentrated into relative density for 1.05~
1.10（50℃）Clear cream, add ethanol, make alcohol content up to 80%, stand 24 it is small when, filtration, decompression filtrate recycling ethanol is simultaneously dense
Relative density is reduced to as 1.10~1.20（80℃）Clear cream, take menthol, sodium benzoate 0.5-1.5g, citric acid 0.5-
1.5g, flavoring pineapple essence 5-6ml, with ethanol 20ml, after stirring and dissolving, add in above-mentioned medicinal extract, add water to 1000ml, stirring is equal
Even, filtration is filling, to obtain the final product.
Open larynx sword spray（Child form）Differentiate and assay is studied
Open larynx sword spray（Child form）Quality standard is now recorded in State Food and Drug Administration national drug standards WS-
10132 (ZD-0132) -2002-2012Z, the approval number of the drug are Chinese medicines quasi-word Z20025142.To improve the drug standards, to place
Square title is revised, and revision has been carried out to existing standard, has been improved；Bicolor thin layer in prescription is differentiated and is revised,
Add using bicolor control medicinal material as control；The thin layer of specification menthol differentiates language description；Add mountain beans in prescription
The thin layer of root differentiates；Separately the thin layer discriminating to cicada slough is studied.Add white to the main component rock of bicolor in prescription
The assay of dish element；The content assaying method of matrine and oxymatrine in subprostrate sophora is revised；
【Differentiate】This product is made of four taste Chinese medicines, and the thin layer for having revised bicolor differentiates, using Bergenin as control in primary standard
Differentiated, test sample preparation method is complicated, and now former method is revised, bicolor control medicinal material is added, simplifies
Test sample preparation method, this method specificity is strong, favorable reproducibility, therefore is included in standard body；Language is differentiated to the thin layer of menthol
Description has carried out specification, and solvent agents useful for same benzene is changed to toluene；The thin layer for adding subprostrate sophora differentiates, with matrine
For control, stablize through investigating this method, favorable reproducibility, therefore be included in standard body.
1st, the thin layer of bicolor differentiates
With reference to《Chinese Pharmacopoeia》The thin layer of one " bicolor " medicinal material of version in 2015 differentiates that method is as follows：This product 2ml is taken, is evaporated,
Residue adds methanol 1ml to make dissolving, as test solution.Bicolor control medicinal material 0.5g separately is taken, adds methanol 20ml, is ultrasonically treated
30 minutes, let cool, filter, filtrate is evaporated, and residue adds methanol 1ml to make dissolving, as control medicinal material solution.Bergenin is taken to compare
Product, add methanol that solution of every 1ml containing 0.5mg is made, as reference substance solution.The negative sample solution 1ml of scarce bicolor is taken again,
It is made in the same way of negative sample solution.According to thin-layered chromatography (《Chinese Pharmacopoeia》Four general rules 0502 of version in 2015) test, in absorption
Each 3 μ l of four kinds of solution are stated, are put respectively on same silica gel g thin-layer plate, with chloroform-acetate-methanol (5:4:2) it is exhibition
Agent is opened, is unfolded, is taken out, is dried, spray with -1% potassium ferricyanide solution (1 of 1% liquor ferri trichloridi:1) mixed solution.As a result,
In test sample chromatography, on position corresponding with reference substance and control medicinal material chromatography, spot and the negative nothing for showing same color are dry
Disturb.
Serviceability test is carried out at the same time, has investigated different manufacturers silica G plate (Haiyang Chemical Plant, Qingdao and Yantai City's chemistry work
Industry research institute) and different expansion temperature（25 DEG C and 8 DEG C）, relative humidity（58% and 75%）Etc. influence factor, as a result this expansion
Condition principal spot is clear, good tolerance.Therefore this thin layer method is included in text.
Therefore the thin-layer identification method of definite bicolor is：This product 2ml is taken, is evaporated, residue adds methanol 1ml to make dissolving, as
Test solution.Bicolor control medicinal material 0.5g separately is taken, adds methanol 20ml, is ultrasonically treated 30 minutes, lets cool, is filtered, filtrate is steamed
Dry, residue adds methanol 1ml to make dissolving, as control medicinal material solution.Bergenin reference substance is taken again, is added methanol that every 1ml is made and is contained
The solution of 0.5mg, as reference substance solution.According to thin-layered chromatography (《Chinese Pharmacopoeia》Four general rules 0502 of version in 2015) experiment,
Above-mentioned each 3 μ l of three kinds of solution are drawn, are put respectively on same silica gel g thin-layer plate, with chloroform-acetate-methanol (5:4:
2) it is solvent, is unfolded, takes out, dry, spray with -1% potassium ferricyanide solution (1 of 1% liquor ferri trichloridi:1) mixed solution.
In test sample chromatography, on position corresponding with control medicinal material and reference substance chromatography, the spot of same color is shown.
2nd, the thin layer of menthol differentiates
Solvent agents useful for same benzene is changed to toluene, method is as follows after change：This product 10ml is taken, is extracted 2 times with ether shaking,
Each 20ml, volatilizees naturally.Residue adds methanol 2ml to make dissolving, as test solution.Menthol reference substance separately is taken, adds methanol
Solution of every 1ml containing 2mg is made, as reference substance solution.According to thin-layered chromatography（《Chinese Pharmacopoeia》Four general rules of version in 2015
0502）Experiment, draws test solution 10 μ l, 5 μ l of reference substance solution, puts respectively on same silica gel g thin-layer plate, with toluene-
Ethyl acetate（19:1）For solvent, it is unfolded, takes out, dry, spray with vanillin-sulfuric acid test solution-ethanol（1:4）, add at 100 DEG C
Heat is to clear spot.As a result：In test sample chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown.
Serviceability test is carried out at the same time, has investigated different manufacturers silica G plate (Haiyang Chemical Plant, Qingdao and Yantai City's chemistry work
Industry research institute) and different expansion temperature（26 DEG C and 7 DEG C）, relative humidity（58% and 79%）Etc. influence factor, as a result this expansion
Condition principal spot is clear, good tolerance.Therefore this thin layer method is included in text.
Therefore the thin-layer identification method of definite menthol is：This product 10ml is taken, is extracted 2 times, each 20ml with ether shaking, from
So volatilization.Residue adds methanol 2ml to make dissolving, as test solution.Menthol reference substance separately is taken, adds methanol that every 1ml is made and contains
The solution of 2mg, as reference substance solution.According to thin-layered chromatography（《Chinese Pharmacopoeia》Four general rules 0502 of version in 2015）Experiment, inhales
10 μ l of test solution, 5 μ l of reference substance solution are taken, is put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate（19:
1）For solvent, it is unfolded, takes out, dry, spray with vanillin-sulfuric acid test solution-ethanol（1:4）, clear spot is heated at 100 DEG C.
In test sample chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown.
3rd, the thin layer of subprostrate sophora differentiates
With reference to《Chinese Pharmacopoeia》The thin layer of one " subprostrate sophora " medicinal material of version in 2015 differentiates that method is as follows：This product 2ml is taken, adds three
Chloromethanes 10ml, strong ammonia solution 0.2ml, shake 15 minutes, and filtration, filtrate is evaporated, and residue adds chloroform 0.5ml to make dissolving,
As test solution.Subprostrate sophora control medicinal material and each 0.5g of medicinal material separately are taken, is made in the same way of control medicinal material and medicinal material solution.Take hardship
Join alkali reference substance, add chloroform that every 1ml respectively mixed solutions containing 1mg are made, as reference substance solution.Scarce subprostrate sophora is taken again
Negative sample solution 2ml is made in the same way of negative sample solution.According to thin-layered chromatography (《Chinese Pharmacopoeia》Four general rules of version in 2015
0502) test, draw each 3 μ l of above-mentioned 4 kinds of solution, 5 μ l of reference substance solution respectively, put respectively on same silica gel g thin-layer plate, with
Chloroform-methanol-strong ammonia solution (4:1:0.1) it is solvent, is unfolded, takes out, dry, spray with dilute bismuth potassium iodide test solution.
As a result：In test sample chromatography, with the position of matrine reference substance, spot and R f value is larger, adjust solvent ratio
Example, is changed to chloroform-methanol-strong ammonia solution (12:1:0.1), while test sample preparation method is changed to subprostrate sophora to contain
Measure and customize standby test solution, be evaporated, point sample.
As a result：In test sample chromatography, on position corresponding with matrine reference substance chromatography, identical orange color spot is shown
Point, and it is negative noiseless.
Serviceability test is carried out at the same time, has investigated different manufacturers silica G plate (Haiyang Chemical Plant, Qingdao and Yantai City's chemistry work
Industry research institute) and different expansion temperature（26 DEG C and 8 DEG C）, relative humidity（61% and 76%）Etc. influence factor, as a result this expansion
Condition good tolerance, principal spot are clear.Therefore this thin layer method is included in text.
The thin-layer identification method for determining subprostrate sophora is：Take [assay] matrine and test sample under oxymatrine item molten
Liquid 5ml, is evaporated, and residue adds chloroform 1ml to make dissolving, as test solution.Matrine reference substance separately is taken, adds chloroform
Solution of every 1ml containing 1mg is made, as reference substance solution.According to thin-layered chromatography (《Chinese Pharmacopoeia》Four general rules of version in 2015
0502) test, draw test solution 3 μ l, 2 μ l of reference substance solution, put respectively on same silica gel g thin-layer plate, with three chloromethanes
Alkane-methanol-strong ammonia solution（12:1:0.1）For solvent, it is unfolded, takes out, dry, spray with dilute bismuth potassium iodide test solution.Test sample color
In spectrum, with reference substance chromatography relevant position, showing identical orange-yellow spot.
3rd, cicada slough-differentiate
Bibliography[1], the components such as amino acid are mainly contained in cicada slough, and through inspection information, amino acid in cicada slough is differentiated,
To determine the species of amino acid, content height, tentatively using liquid phase mirror method for distinguishing, 17 kinds of amino acid are screened, method
It is as follows：
Chromatographic condition chromatographic column：113#Kromasil100-5C18 (4.6 × 250mm, 5 μm) mobile phase：30mM sodium acetates（36%
Acetic acid tune pH to 6.50）：Acetonitrile 355：14 be mobile phase A, and 80% acetonitrile is Mobile phase B, and according to the form below carries out gradient elution：Column temperature
40℃；Flow velocity 1.0ml/min；5 μ l of sample size；Detection wavelength 254nm.
Deriving method, accurate 100 μ l of mixed reference substance solution, the 400 μ l of test solution of measuring is in 10ml centrifuge tubes respectively
In, add 0.1mol/LHCl and supply 1ml, vortex mixed uniformly adds 0.1mol/LPITC acetonitrile solutions 0.5ml, 1.0mol/ afterwards
L triethylamine acetonitrile solution 0.5ml, vortex mixed is uniform, room temperature place 1 it is small when after, add 4ml n-hexanes, vortex mixed is uniform,
Room temperature is placed after ten minutes, draws lower floor's solution, to obtain the final product.See Figure 13.
Each sequence number refers to respectively in Figure 13：1st, L-aminobutanedioic acid 2, glutamic acid 3, serine 4, glycine 5, histidine 6, arginine
7th, threonine
8th, alanine 9, proline 10, tyrosine 11, valine 12, methionine 13, cystine
14th, isoleucine 15, leucine 16, phenylalanine 17, lysine
As a result：Shown according to above collection of illustrative plates, the higher amino acid of content mainly there are following 3 kinds in sample：Arginine, proline,
Leucine, although there are chromatographic peak in other amino acid corresponding positions, but peak area is smaller, and content is relatively low.
According to the above results, after preparing arginine, proline, leucine reference substance solution and negative sample solution, sample introduction,
The result is shown in Figure 14.
It is followed successively by from top to bottom in Figure 14：Leucine；Arginine, proline, leucine mixing control；Sample；It is negative
As a result：Understand from the graph, with arginine, proline position, feminine gender has interference, consider due to being gradient elution, hold
Appearance time drift is easily caused, therefore chromatographic condition is optimized, mobile phase ratio is adjusted to 80：20.The result is shown in Figure 15.
It is from secondary in Figure 15：Sample；It is negative；Arginine, proline, leucine mixing control；
It is followed successively by from top to bottom in Figure 16：Sample；It is negative；Leucine
As a result：More than it was found from two figures, main chromatographic peak is basically identical in negative sample and sample chromatogram figure.
【Assay】The assay of Bergenin in bicolor is added, Coumarins is mainly contained in bicolor
Studying point, its assay is more using Bergenin as index components,《Chinese Pharmacopoeia》One " bicolor " the medicinal material item of version in 2015
Under establish the assay of Bergenin, therefore establish the assay using Bergenin as index with reference to official method,
Methodology validation the results show the method is stablized, and durability is good.The assay of matrine in primary standard is revised, because of primary standard
Method can only extract matrine, in order to ensure the accuracy and integrality of sample measure, its method be revised, after revision
Method can ensure that matrine and oxymatrine extraction are complete in preparation, and using the total amount of matrine and oxymatrine as finger
Mark, has carried out assay.
Bergenin shines liquid chromatography（《Chinese Pharmacopoeia》Four general rules 0512 of version in 2015）Measure.
Instrument and reagent
Liquid chromatograph Shimadzu LC-2010Cht；
Chromatographic column Inertsil ODS-3（4.6×250mm 5μm）；
Electronic analytical balance SARTORIUS BT25S（Ten a ten thousandths）；
Electronic analytical balance SARTORIUS BS124S（A ten thousandth）；
Research sample：Sanli Pharmaceutical Co., Ltd., Guizhou produces, specification 15ml（Lot number：20160202、20150959、
20150971）, 20ml（Lot number：20150501）, 30ml（Lot number：20150883、20150880、20150801、20150889、
20150856、20150950、20150958、20150964、20150920、20150930、20150937）.Wherein 20160202
Batch be used for content assaying method grope and content assaying method verification.
Reagent：Methanol is chromatographically pure, and DIKMA companies produce；Water is Wahaha Pure Water.
Bergenin reference substance（Lot number：MUST-16041803, purity 99.87%）, purchased from Chengdu Man Site biotechnologies
Co., Ltd.
The selection of chromatographic condition
1.2.1 the selection of Detection wavelength
Take Bergenin reference substance appropriate, it is accurately weighed, add methanol that the reference substance solution of every 1ml 50 μ g containing Bergenin is made,
It is scanned under 200~400nm wavelength, it is seen that Bergenin has maximum absorption band at 272nm, with《Chinese Pharmacopoeia》2015
Determination of Bergenin measure Detection wavelength 275nm is not much different under year one " bicolor " item of version, therefore selects 275nm as detection ripple
It is long.（See Figure 17）
1.2.2 the selection of mobile phase
Reference《Chinese Pharmacopoeia》Content assaying method under one " bicolor " item of version in 2015, mobile phase use methanol：Water（25:
75）, as a result negative sample have interference, adjustment mobile phase ratio is methanol-water（23:77）, results sample peak peak shape, separating degree are equal
Well.Therefore determine that mobile phase is methanol-water（23:77）.
Prepared by test solution investigates
1.3.1 dilute solvent selection
Precision measures this product 2ml, puts in 10ml measuring bottles, is separately added into methanol, water, appropriate Diluted Alcohol, is ultrasonically treated（Power
250W, frequency 40kHz）30 minutes, letting cool, be diluted to scale with coordinative solvent, shake up, filter, precision draws 5 μ l of subsequent filtrate,
Inject liquid chromatograph, measure, calculate, to obtain the final product.It the results are shown in Table 1.
Table 1 extracts solvent and investigates
| Extract solvent | Determination of Bergenin（mg/ml） |
| Methanol | 0.4014 |
| Water | 0.3932 |
| Diluted Alcohol | 0.3920 |
The result shows that respectively the result difference of extraction solvent is little, methanol extraction easily filtration, therefore it is solvent to select methanol.
The investigation of extracting mode
Directly dilute sample introduction sample：Precision measures this product 2ml, puts 10ml measuring bottles, adds methanol to shake up, filter to scale；
Ultrasound, shaking sample：Precision measures this product 2ml, puts in 10ml measuring bottles, adds methanol appropriate, be ultrasonically treated respectively（Power
250W, frequency 40kHz）15 minutes, shaking 30 minutes, let cool, with methanol dilution to scale, shake up, filter；It is accurate respectively to draw
5 μ l of subsequent filtrate, inject liquid chromatograph, measure, and calculate, to obtain the final product.It the results are shown in Table 2.
The investigation of 2 extracting mode of table
| Extracting mode | Determination of Bergenin（mg/ml） |
| Ultrasound | 0.4028 |
| Shaking | 0.4016 |
| Directly dilute sample introduction | 0.4047 |
The result shows that the content difference of three kinds of extracting modes is little, therefore directly dilute sample introduction.
Content assaying method primarily determines that
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；With methanol-water（23:77）For
Mobile phase；Detection wavelength is 275nm.Number of theoretical plate is calculated by Bergenin peak should be not less than 1500.
The preparation of reference substance solution takes Bergenin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made and contains purple bergenia herb
The solution of 80 μ g of element, to obtain the final product.
The preparation of test solution is accurate to measure this product 2ml, puts in 10ml measuring bottles, adds methanol to shake up, filter to scale,
Take subsequent filtrate, to obtain the final product.
Determination method difference is accurate to draw reference substance solution and each 5 μ l of test solution, injects liquid chromatograph, measures, i.e.,
.
Methodological study
1.5.1 specificity is tested
To investigate the measure whether other flavour of a drug and auxiliary material disturb out Bergenin in larynx sword spray, medicine is weighed in prescription ratio
Taste and auxiliary material are made in the same way of the negative sample of scarce bicolor, and the negative sample of the scarce bicolor of processing method preparation according to test sample
Product solution sample introduction, records chromatogram（See Figure 18）.The result shows that do not have in negative chromatography in retention time corresponding with Bergenin
There is chromatographic peak, show that other flavour of a drug beyond bicolor and auxiliary material are noiseless to the measure of Bergenin, finished product is measured with this law
The content of middle Bergenin has specificity.
1.5.2 peak purity is investigated
Reference substance and test solution are prepared by fixed reference substance and test sample preparation method, by having determined that chromatographic condition adopts
Full wavelength scanner is carried out with PDA detectors, peak purity is calculated, is as a result not detected by impurity peaks；Peak purity index is 1.0000, is said
It is bright under the chromatographic condition, Bergenin chromatography peak purity meets regulation.（See Figure 19）
1.5.3 the investigation of linear relationship
Precision weighs Bergenin reference substance（Lot number：MUST-16041803, purity 99.87%）13.69mg, puts 50ml measuring bottles
In, add methanol to make dissolving and be diluted to scale, shake up, it is accurate respectively up to the reference substance storing solution that concentration is 0.2738mg/ml
Stock solution 0.5ml, 1ml, 2ml, 3ml, 5ml are measured, is split in 10ml measuring bottles, with methanol dilution to scale, is shaken up, be configured to
Concentration is respectively that 13.69 μ g/ml, 27.38 μ g/ml, 54.76 μ g/ml, 82.14 μ g/ml, the reference substance of 136.90 μ g/ml are molten
Liquid.It is accurate respectively to draw above-mentioned reference substance solution together with storing solution（273.80 μ g/ml of concentration）5 μ l, inject liquid chromatograph, shine
Above-mentioned liquid phase chromatogram condition is measured.Using sample size as abscissa, peak area is ordinate, draws standard curve.Calculate back
Return equation：
Bergenin Y=1217374.4058X-3792.6377, related coefficient：r=1.0000
The result shows that Bergenin sample size sample size and peak area linear relationship between the μ g of 0.0685 μ g~1.3690 is good
It is good, it is shown in Table 3, Figure 20.
3 Bergenin linear relationship of table
| Sample size μ g | Peak area |
| 0.0685 | 80061 |
| 0.1369 | 164002 |
| 0.2738 | 332093 |
| 0.4107 | 498443 |
| 0.6845 | 819301 |
| 1.3690 | 1666564 |
1.5.4 instrument precision experiment precision takes this product（Lot number：20160202）2ml, by text test solution
Prepare and operated under determination method item, continuous sample introduction 6 times, measurement result is shown in Table 4.
4 instrument precision result of the test of table（n=6）
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | Average value | RSD（%） |
| Peak area | 476105 | 475944 | 478931 | 478011 | 477980 | 477958 | 477488 | 0.25 |
The result shows that instrument precision is good.
Stability test precision takes this product（Lot number：20160202）2ml, by the preparation and survey of text test solution
Determine to operate under method item, sample introduction measures when placement 0,2,4,8,12,24 is small after preparation respectively, and measurement result is shown in Table 5.
5 stability test result of table
| Time（h） | 0 | 2 | 4 | 8 | 12 | 24 | Average value | RSD（%） |
| Peak area | 476105 | 479091 | 480127 | 476470 | 474336 | 471883 | 476335 | 0.64 |
The result shows that test solution internal stability when 24 is small is good.
Repetitive test experimenter（A）Operation, precision take this product（Lot number：20160202）2ml, totally 6 parts, by just
Literary test solution is prepared and operated under determination method item.6 are the results are shown in Table, attached drawing 14 to 20.
6 repetitive test result of table（n=6）
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | Average value | RSD（%） |
| Determination of Bergenin（mg/ml） | 0.4001 | 0.4016 | 0.4037 | 0.4045 | 0.4058 | 0.4024 | 0.4030 | 0.51 |
The result shows that RSD<2.0%, illustrate that the repeatability of this method is good.
Intermediate precision experiment selects different minutes, different high performance liquid chromatographs, different experimenters
（B）, it is accurate to take this product（Lot number：20160202）2ml, is prepared by text test solution and is grasped under determination method item
Make.It the results are shown in Table 7.
7 Intermediate precision result of the test of table（n=6）
As a result, Bergenin average content is 0.3973mg/ml, RSD in the sample of A and B measure<3.0%.Show, in this method
Between precision it is good.
Accuracy testing
Using sample-adding absorption method.Precision weighs Bergenin reference substance（Lot number is：MUST-16041803, purity 99.87%）
10.00mg, puts in 50ml measuring bottles, adds methanol to shake up to scale, the reference substance solution that concentration is 0.2000mg/ml is made.It is accurate
Measure this product 1ml（Totally 9 parts）, put in 10ml measuring bottles, it is accurate respectively to add reference substance solution 1ml, 2ml, the 3ml configured（It is flat
3 parts of row）, add methanol to shake up to scale, take subsequent filtrate, to obtain the final product.It is shown in Table 8.
By repetitive test derived sample（Lot number：20160202）Middle content is 0.4030mg/ml.
8 accuracy testing of table
| Title | Sampling amount（ml） | Measured amount（mg） | Content in test sample（mg） | Reference substance addition（mg） | The rate of recovery（%） |
| 1 | 1 | 0.6073 | 0.4030 | 0.2000 | 102.15 |
| 2 | 1 | 0.6124 | 0.4030 | 0.2000 | 104.70 |
| 3 | 1 | 0.6119 | 0.4030 | 0.2000 | 104.45 |
| 4 | 1 | 0.8056 | 0.4030 | 0.4000 | 100.65 |
| 5 | 1 | 0.8135 | 0.4030 | 0.4000 | 102.63 |
| 6 | 1 | 0.8138 | 0.4030 | 0.4000 | 102.70 |
| 7 | 1 | 1.0284 | 0.4030 | 0.6000 | 104.23 |
| 8 | 1 | 1.0119 | 0.4030 | 0.6000 | 101.48 |
| 9 | 1 | 1.0204 | 0.4030 | 0.6000 | 102.90 |
The result shows that average recovery rate 102.88%, RSD 1.34%, show that this method accuracy is good.
Serviceability test investigates different chromatographic columns, different column temperatures, different in flow rate, different mobile phase ratios to this chromatographic condition
Durability, the results are shown in Table 9, Figure 21-24.
9 serviceability test of table
The result shows that measurement result is basically identical under the conditions of each, RSD<3%, Bergenin peak type is sharp, symmetrical in chromatogram,
Separating degree is good, shows this method difference chromatographic column, different column temperature, different in flow rate, different mobile phase ratio good tolerances.
Result of study experiment above shows, opens larynx sword spray（Child form）The content assaying method of middle Bergenin is special
Attribute is strong, and stability, repeatability, Intermediate precision, accuracy meet regulation, and the durability of chromatographic condition is preferable, therefore
This liquid phase chromatogram condition can be used for out larynx sword spray（Child form）The assay of middle Bergenin.
Sample size measures
15 batches of preparations are carried out with the assay of Bergenin by the preparation and determination method of test solution identified above.As a result
It is shown in Table 10.
The assay result of 10 15 batches of preparation Bergenins of table
| Lot number | Content（mg/ml） | The rate of transform（%） |
| 20160202 | 0.4167 | |
| 20150964 | 0.5269 | |
| 20150971 | 0.5988 | |
| 20150959 | 0.5880 | |
| 20150958 | 0.4682 | |
| 20150950 | 0.6196 | |
| 20150937 | 0.4071 | |
| 20150930 | 0.6442 | |
| 20150920 | 0.5299 | |
| 20150889 | 0.4646 | |
| 20150883 | 0.3891 | |
| 20150880 | 0.4844 | |
| 20150856 | 0.5224 | |
| 20150801 | 0.3957 | |
| 20150501 | 0.7768 |
Conclusion:The measurement result of Bergenin is * * * mg/ml in 15 batches of samples, with the Determination of Bergenin in bicolor medicine materical crude slice
Not less than pharmacopeic limits（1.0%）For foundation, at the same time, it is contemplated that various influence factors in actual production process etc., fix tentatively this product
Per ml containing bicolor with Bergenin (C14H16O9) meter, 0.25mg must not be less than.（It is equivalent to the preparation rate of transform）.
Matrine and oxymatrine shine liquid chromatography（《Chinese Pharmacopoeia》Four general rules 0512 of version in 2015）Measure.
Instrument and reagent
Liquid chromatograph Shimadzu LC-2010Cht；
Chromatographic column Inertsil NH2 (5 μm of 4.6*250mm)；
Electronic analytical balance SARTORIUS BT25S（Ten a ten thousandths）；
Electronic analytical balance SARTORIUS BS124S（A ten thousandth）；
Research sample:Sanli Pharmaceutical Co., Ltd., Guizhou produces, specification 15ml（Lot number:20160202、20150959、
20150971）, 20ml（Lot number:20150501）, 30ml（Lot number:20150883、20150880、20150801、20150889、
20150856、20150950、20150958、20150964、20150920、20150930、20150937）.Wherein 20160202
Batch be used for content assaying method grope and content assaying method verification.
Reagent:Acetonitrile, isopropanol are chromatographically pure, and DIKMA companies produce；Water is Wahaha Pure Water.
Matrine reference substance（Lot number:110805-200508）；Oxymatrine reference substance（Lot number:110780-201508
Purity 92.5%）Purchased from National Institute for Food and Drugs Control.
The selection of chromatographic condition
2.2.1 the selection of Detection wavelength
Take matrine, oxymatrine reference substance appropriate, it is accurately weighed, add acetonitrile-absolute ethyl alcohol（80:20）Every 1ml is made to contain
The reference substance solution of matrine 0.1mg, oxymatrine 0.075mg, are scanned, it is seen that kuh-seng under 200~400nm wavelength
Alkali, oxymatrine have maximum absorption band at 202nm,《Chinese Pharmacopoeia》Kuh-seng under one " subprostrate sophora " item of version in 2015
Alkali, oxymatrine concentration measure Detection wavelength are 210nm, consider to absorb close to end under 202nm, impurity is more, therefore refers to
It is Detection wavelength that pharmacopeia condition, which selects 210nm,.（See Figure 25）
2.2.2 the selection of mobile phase
With reference to primary standard mobile phase acetonitrile-phosphoric acid solution of absolute ethyl alcohol -0.3%（80:10:10）, as a result, separating degree is bad；Reference
《Chinese Pharmacopoeia》Content assaying method under one " subprostrate sophora " item of version in 2015, it is molten using mobile phase acetonitrile-phosphoric acid of isopropanol -3%
Liquid（80:5:15）, as a result negative sample have interference, adjustment mobile phase ratio is acetonitrile-phosphoric acid solution of isopropanol -3%（85:5:
10）, results sample peak peak shape, separating degree are good.Therefore determine that mobile phase is acetonitrile-phosphoric acid solution of isopropanol -3%（85:5:
10）.
Prepared by test solution investigates
2.3.1 neutral alumina dosage is investigated
Precision measures this product 2ml, adds 2g neutral aluminas（100-200 mesh）Mix thoroughly, put 70 DEG C of water bath methods, be added in neutral oxygen
Change aluminium column（100-200 mesh, 1g, 2g, 3g, 4g, internal diameter 1.5cm）On, successively with chloroform, chloroform:Methanol（7:3）Respectively
30ml is eluted, and is collected eluent, is put 70 DEG C of water bath methods, residue adds acetonitrile-absolute ethyl alcohol（80:20）Dissolving, is transferred to 10ml
In measuring bottle, with acetonitrile-absolute ethyl alcohol（80:20）Scale is settled to, is shaken up, miillpore filter filtration, to obtain the final product.It the results are shown in Table 11.
11 neutral alumina dosage of table is investigated
| Neutral alumina dosage | Matrine and Oxymatrine total alkali content（mg/ml） |
| 3g | 0.4474 |
| 4g | 0.4530 |
| 5g | 0.4659 |
| 6g | 0.2368 |
The result shows that neutral alumina dosage, in 3g, 4g, 5g, content difference is little, but sample impurity is more during 3g, together
When for save resource, select neutral alumina dosage be 4g.
Eluting solvent is investigated
Precision measures this product 2ml, adds 2g neutral aluminas（100-200 mesh）Mix thoroughly, put 70 DEG C of water bath methods, be added in neutral oxygen
Change aluminium column（100-200 mesh, 2g, internal diameter 1.5cm）On, sample is 1. successively with dichloromethane, dichloromethane:Methanol（7:3）Respectively
20ml is eluted；Sample is 2. successively with chloroform, chloroform:Methanol（7:3）Each 20ml elutions；Sample 3. successively with ether,
Ether:Methanol（7:3）Each 20ml elutions, collect eluent, put 70 DEG C of water bath methods, residue adds acetonitrile-absolute ethyl alcohol（80:20）
Dissolving, is transferred in 10ml measuring bottles, with acetonitrile-absolute ethyl alcohol（80:20）Scale is settled to, is shaken up, miillpore filter filtration, i.e.,
.It the results are shown in Table 12.
12 eluting solvent of table is investigated
| Eluting solvent | Matrine and Oxymatrine total alkali content（mg/ml） |
| Dichloromethane, dichloromethane:Methanol（7:3） | 0.4578 |
| Chloroform, chloroform:Methanol（7:3） | 0.4774 |
| Ether, ether:Methanol（7:3） | 0.4641 |
The result shows that chloroform, chloroform:Methanol（7:3）Effect is preferable during elution.
Amount of eluent is investigated
Precision measures this product 2ml, adds 2g neutral aluminas（100-200 mesh）Mix thoroughly, put 70 DEG C of water bath methods, be added in neutral oxygen
Change aluminium column（100-200 mesh, 2g, internal diameter 1.5cm）On, successively with chloroform, chloroform:Methanol（7:3）Each 10ml,
20ml, 30ml are eluted, and are collected eluent, are put 70 DEG C of water bath methods, residue adds acetonitrile-absolute ethyl alcohol（80:20）Dissolving, is transferred to
In 10ml measuring bottles, with acetonitrile-absolute ethyl alcohol（80:20）Scale is settled to, is shaken up, miillpore filter filtration, to obtain the final product.It the results are shown in Table
13。
13 amount of eluent of table is investigated
| Amount of eluent | Matrine and Oxymatrine total alkali content（mg/ml） |
| 10ml | 0.4385 |
| 20ml | 0.4770 |
| 30ml | 0.4669 |
The result shows that active ingredient can be eluted during 20ml complete.
Content assaying method primarily determines that
Chromatographic condition and system suitability are using amino bonded silica gel as filler, with acetonitrile-phosphoric acid solution of isopropanol -3%
(85:5:10) it is mobile phase；Detection wavelength is 210nm.
The preparation of reference substance solution takes matrine reference substance, oxymatrine reference substance appropriate, accurately weighed, add acetonitrile-
Absolute ethyl alcohol（80:20）It is respectively prepared every 1ml 0.1mg containing matrine, the mixed solution of oxymatrine 0.075mg, to obtain the final product.
The preparation of test solution is accurate to measure this product 2ml, adds 2g neutral aluminas（100-200 mesh）Mix thoroughly, put 70
DEG C water bath method, is added in neutral alumina column（100-200 mesh, 2g, internal diameter 1.5cm）On, successively with chloroform, three chloromethanes
Alkane:Methanol（7:3）Each 20ml elutions, collect eluent, put 70 DEG C of water bath methods, residue adds acetonitrile-absolute ethyl alcohol（80:20）It is molten
Solution, is transferred in 10ml measuring bottles, adds acetonitrile-absolute ethyl alcohol（80:20）To scale, shake up, filter, take subsequent filtrate, to obtain the final product.
Determination method difference is accurate to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures, i.e.,
.
Methodological study
2.5.1 specificity is tested
To investigate whether other flavour of a drug and auxiliary material disturb out the measure of matrine and oxymatrine in larynx sword spray, by prescription
Ratio weighs flavour of a drug and auxiliary material is made in the same way of the negative sample of scarce subprostrate sophora, and prepares scarce mountain beans according to the processing method of test sample
The negative sample solution sample introduction of root, records chromatogram（See Figure 26）.The result shows that bitter with matrine, oxidation in negative chromatography
The ginseng corresponding retention time of alkali does not have chromatographic peak, shows other flavour of a drug beyond subprostrate sophora and auxiliary material to matrine, Oxymatrine
The measure of alkali is noiseless, measures matrine in finished product with this law, the content of oxymatrine has specificity.
2.5.2 peak purity is investigated
Reference substance and test solution are prepared by fixed reference substance and test sample preparation method, by having determined that chromatographic condition adopts
Full wavelength scanner is carried out with PDA detectors, peak purity is calculated, is as a result not detected by impurity peaks；Peak purity index is 0.999996,
Illustrate under the chromatographic condition, matrine chromatography peak purity meets regulation.Oxymatrine cannot be shown due to not there is peak
Peak purity.（See Figure 27）
2.5.3 the investigation of linear relationship
2.5.3.1 matrine
Precision weighs matrine reference substance（Lot number is:110805-200508）11.13mg, puts in 10ml measuring bottles, with acetonitrile-nothing
Water-ethanol（80:20）Make dissolving and be diluted to scale, shake up, be the reference substance storing solution of 1.113mg/ml up to concentration, then divide
Inaccurate measurement above-mentioned storing solution 1ml, 2ml, 5ml are split in 10ml measuring bottles, with acetonitrile-absolute ethyl alcohol（80:20）It is diluted to quarter
Degree, shakes up, up to the reference substance solution that concentration is 0.1113mg/ml, 0.2226mg/ml, 0.5565mg/ml, then accurate measurement
Concentration is that reference substance solution 1ml, 2ml, 5ml of 0.1113mg/ml are split in 10ml measuring bottles, with acetonitrile-absolute ethyl alcohol（80:20）
Scale is diluted to, is shaken up, up to the reference substance solution that concentration is 0.0111mg/ml, 0.0223mg/ml, 0.0557mg/ml, is divided
Not it is accurate draw concentration for 0.01113mg/ml, 0.02226mg/ml, 0.05565mg/ml, 0.11130mg/ml,
Each 10 μ l of reference substance solution of 0.22260mg/ml, 0.55650mg/ml, inject liquid chromatograph, according to above-mentioned liquid phase chromatogram condition
It is measured.Using sample size as abscissa, peak area is ordinate, draws standard curve.
Calculate regression equation:
Y=1540326.3119X+37290.3285, related coefficient:r=1.0000
The result shows that matrine sample size sample size and peak area linear relationship between the μ g of 0.1113 μ g~5.5650 is good,
It is shown in Table 14, Figure 28.
14 matrine linear relationship of table
| Sample size μ g | Peak area |
| 0.1113 | 181948 |
| 0.2226 | 351558 |
| 0.5565 | 884890 |
| 1.1130 | 1825585 |
| 2.2260 | 3471412 |
| 5.5650 | 8594921 |
2.5.3.2 oxymatrine
Precision weighs oxymatrine reference substance（Lot number is:110780-201508, purity 92.50%）10.32mg put 25ml
In measuring bottle, add acetonitrile-absolute ethyl alcohol（80:20）Dissolve and be diluted to scale, shake up, up to pair that concentration is 381.84 μ g/ml
According to product storing solution.It is accurate respectively to measure stock solution 2ml, 3ml, 5ml, split in 10ml measuring bottles, with acetonitrile-absolute ethyl alcohol（80:
20）Be diluted to scale, shake up, up to concentration for 76.37 μ g/ml, 114.55 μ g/ml, 190.92 μ g/ml reference substance solution,
Shake up；It is accurate respectively again to measure reference substance solution 2ml, 5ml that concentration is 76.37 μ g/ml, put in 10ml measuring bottles, with acetonitrile-nothing
Water-ethanol（80:20）Scale is diluted to, is shaken up, up to the reference substance solution that concentration is 15.27 μ g/ml, 38.18 μ g/ml, difference
Precision draws concentration as 15.27 μ g/ml, 38.18 μ g/ml, 76.37 μ g/ml, 114.55 μ g/ml, 190.92 μ g/ml, 381.84
The 10 μ l of reference substance solution of μ g/ml, inject liquid chromatograph, are measured according to above-mentioned liquid phase chromatogram condition.Using sample size as horizontal stroke
Coordinate, peak area are ordinate, draw standard curve.
Calculate regression equation:
Y=1574260.1967X+8464.1091, related coefficient:r=0.9998
The result shows that oxymatrine sample size sample size and peak area linear relationship between the μ g of 0.1527 μ g~3.8184 is good
It is good, it is shown in Table 15, Figure 29.
15 oxymatrine linear relationship of table
| Sample size μ g | Peak area |
| 0.1527 | 237830 |
| 0.3818 | 603369 |
| 0.7637 | 1184356 |
| 1.1455 | 1804742 |
| 1.9092 | 3098318 |
| 3.8184 | 5985922 |
2.5.4 instrument precision experiment takes this product（Lot number:20160202）, preparation and determination method by text test solution
Operation, continuous sample introduction 6 times, measurement result are shown in Table 16 under.
16 instrument precision result of the test of table（n=6）
The result shows that instrument precision is good.
Stability test takes this product（Lot number:20160202）, by being grasped under the preparation of text test solution and determination method item
Make, sample introduction measures when placement 0,2,4,8,12,24 is small after preparation respectively, and measurement result is shown in Table 17.
17 stability test result of table（n=6）
The result shows that test solution internal stability when 24 is small is good.
Repetitive test experimenter（A）Operation, takes this product（Lot number:20160202）, it is totally 6 parts, molten by text test sample
Liquid is prepared and operated under determination method item.It the results are shown in Table 18.
18 repetitive test result of table（n=6）
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | Average value | RSD（%） |
| Matrine（mg/ml） | 0.4697 | 0.4708 | 0.4772 | 0.4703 | 0.5038 | 0.4770 | 0.4781 | 2.72 |
| Oxymatrine（mg/ml） | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
The result shows that RSD<3.0%, illustrate that the repeatability of this method is good.
Intermediate precision experiment selects different minutes, different high performance liquid chromatographs, different experimenters
（B）, take this product（Lot number:20160202）, prepare by text test solution and operated under determination method item.It the results are shown in Table
19。
19 Intermediate precision result of the test of table（n=6）
As a result, matrine, oxymatrine average content are 0.4773mg/ml, RSD in the sample of A and B measure<3.0%.Table
Bright, this method Intermediate precision is good.
Accuracy testing
Using sample-adding absorption method.Precision weighs matrine reference substance（Lot number is:110805-200508）12.53mg, puts 50ml amounts
In bottle, add acetonitrile-absolute ethyl alcohol（80:20）Scale is diluted to, is shaken up, the reference substance solution that concentration is 0.2506mg/ml is made.
Precision weighs oxymatrine reference substance again（Lot number is:110780-201508 purity 92.5%）10.25mg, puts 25ml measuring bottles
In, add acetonitrile-absolute ethyl alcohol（80:20）To scale, shake up, the reference substance solution that concentration is 0.3793mg/ml is made.Precision amount
Take this product 1ml（Totally 9 parts）, put in evaporating dish, it is accurate respectively to add the matrine configured, oxymatrine reference substance solution
1ml、2ml、3ml（Parallel 3 parts）, add 2g neutral aluminas（100-200 mesh）Mix thoroughly, put 70 DEG C of water bath methods, be added in neutrality
Alumina column（100-200 mesh, 2g, internal diameter 1.5cm）On, successively with chloroform, chloroform:Methanol（7:3）Each 30ml is washed
It is de-, eluent is collected, puts 70 DEG C of water bath methods, residue adds acetonitrile-absolute ethyl alcohol（80:20）Dissolving, is transferred in 10ml measuring bottles,
Add acetonitrile-absolute ethyl alcohol（80:20）To scale, shake up, filter, take subsequent filtrate, to obtain the final product.It is shown in Table 20,21.
By repetitive test derived sample（Lot number：20160202）Middle matrine content is 0.4781mg/ml, Oxymatrine
Alkali content is 0.
20 matrine accuracy testing of table
| Title | Sampling amount（ml） | Measured amount（mg） | Content in test sample（mg） | Reference substance addition（mg） | The rate of recovery（%） |
| 1 | 1 | 0.7077 | 0.4781 | 0.2506 | 91.62 |
| 2 | 1 | 0.7089 | 0.4781 | 0.2506 | 92.10 |
| 3 | 1 | 0.7136 | 0.4781 | 0.2506 | 93.97 |
| 4 | 1 | 0.9698 | 0.4781 | 0.5012 | 98.10 |
| 5 | 1 | 0.9408 | 0.4781 | 0.5012 | 92.32 |
| 6 | 1 | 0.9407 | 0.4781 | 0.5012 | 92.30 |
| 7 | 1 | 1.1944 | 0.4781 | 0.7518 | 95.28 |
| 8 | 1 | 1.1625 | 0.4781 | 0.7518 | 91.03 |
| 9 | 1 | 1.1894 | 0.4781 | 0.7518 | 94.61 |
21 oxymatrine accuracy testing of table
| Title | Sampling amount（ml） | Measured amount（mg） | Content in test sample（mg） | Reference substance addition（mg） | The rate of recovery（%） |
| 1 | 1 | 0.3614 | 0 | 0.3793 | 95.28 |
| 2 | 1 | 0.3623 | 0 | 0.3793 | 95.52 |
| 3 | 1 | 0.3625 | 0 | 0.3793 | 95.58 |
| 4 | 1 | 0.7378 | 0 | 0.7585 | 97.27 |
| 5 | 1 | 0.7133 | 0 | 0.7585 | 94.04 |
| 6 | 1 | 0.7158 | 0 | 0.7585 | 94.37 |
| 7 | 1 | 1.0945 | 0 | 1.1378 | 96.19 |
| 8 | 1 | 1.0886 | 0 | 1.1378 | 95.68 |
| 9 | 1 | 1.0876 | 0 | 1.1378 | 95.59 |
As a result, matrine average recovery rate is 93.48%, RSD 2.40%, oxymatrine average recovery rate is 95.50%, RSD
For 0.99%, show that this method accuracy is good.
Serviceability test investigates different chromatographic columns, different in flow rate, different mobile phase ratios to the durability of this chromatographic condition,
22 are the results are shown in Table, Figure 30-32.
22 serviceability test of table
The result shows that measurement result is basically identical under the conditions of each, RSD<3%, matrine, oxymatrine peak type in chromatogram
Sharply, symmetrically, separating degree is good, shows this method difference chromatographic column, different in flow rate, different mobile phase ratio good tolerances.
Result of study experiment above shows, opens larynx sword spray（Child form）The content of middle matrine, oxymatrine
Assay method specificity is strong, stability, repeatability, Intermediate precision, accuracy
Deng meeting regulation, and the durability of chromatographic condition is preferable, so liquid phase chromatogram condition can be used for out larynx sword spray
（Child form）The assay of middle matrine, oxymatrine.
Sample size measures
15 batches of preparations progress matrines and oxymatrine are contained by the preparation and determination method of test solution identified above
It is fixed to measure.It the results are shown in Table 23.
The assay result of 23 15 batches of preparation matrines of table and oxymatrine
| Lot number | Content（mg/ml） | The rate of transform（%） |
| 20150920 | 0.3356 | |
| 20150964 | 0.2983 | |
| 20150930 | 0.3378 | |
| 20150950 | 0.3578 | |
| 20150958 | 0.2799 | |
| 20160202 | 0.4781 | |
| 20150889 | 0.2937 | |
| 20150880 | 0.3164 | |
| 20150959 | 0.3316 | |
| 20150801 | 0.2147 | |
| 20150937 | 0.4705 | |
| 20150501 | 0.2300 | |
| 20150856 | 0.2741 | |
| 20150971 | 0.3559 | |
| 20150883 | 0.2602 |
The description of such scheme is the invention for being understood that and using for ease of those skilled in the art, is familiar with ability
The personnel of field technique obviously easily can make embodiment various modifications, therefore, the invention is not restricted to above-mentioned real scheme,
Those skilled in the art's the method according to the invention, does not depart from improvement that scope made and modification all should be in this hair
Within bright protection domain.
Claims (8)
1. a kind of children open larynx sword spray, it is characterised in that：Calculate by weight, it includes 200-300 parts of Crisped Ardisia Ardisia gigantifolia, mountain beans
0.5-2 parts of 200-300 parts of root, 150-250 parts of cicada slough and menthol.
2. a kind of children open the preparation method of larynx sword spray, it is characterised in that：In addition to menthol, by Crisped Ardisia Ardisia gigantifolia, subprostrate sophora
Added water to cook with three taste of cicada slough it is secondary, for the first time 2 it is small when, second 1 it is small when, collecting decoction, filtration, filtrate is concentrated into relatively close
Spend for 1.05~1.10（50℃）Clear cream, add ethanol, make alcohol content up to 80%, stand 24 it is small when, filtration, filtrate decompression is returned
Receive ethanol and be concentrated into relative density as 1.10~1.20（80℃）Clear cream, take menthol, sodium benzoate 0.5-1.5g, citron
Sour 0.5-1.5g, flavoring pineapple essence 5-6ml, with ethanol 20ml, after stirring and dissolving, add in above-mentioned medicinal extract, add water to 1000ml, stir
Mix uniformly, filtration is filling, to obtain the final product.
3. a kind of children open the quality standard method of inspection of larynx sword spray, it is characterised in that：The children open larynx sword spray by
Crisped Ardisia Ardisia gigantifolia, subprostrate sophora, cicada slough and four taste Chinese medicine of menthol composition, differentiate specific as follows：
1）, bicolor thin layer differentiate：Take this product2ml, it is evaporated, residue adds methanol1mlMake dissolving, as test solution；Separately
TakeBicolor control medicinal material 0.5g, adds methanol 20ml, is ultrasonically treated 30 minutes, lets cool, and filters, and filtrate is evaporated, and residue adds methanol 1ml makes dissolving, as control medicinal material solution；Take againBergenin reference substance, adds methanol that solution of every 1ml containing 0.5mg is made；According to
Thin-layered chromatography is tested, and draws above-mentioned each 3 μ l of three kinds of solution, is put respectively on same silica gel g thin-layer plate, with chloroform-second
Acetoacetic ester-methanol (5:4:2) it is solvent, is unfolded, takes out, dry, spray with -1% potassium ferricyanide solution of 1% liquor ferri trichloridi
(1:1) mixed solution；In test sample chromatography, on position corresponding with control medicinal material and reference substance chromatography, same color is shown
Spot；
2）The thin layer of menthol differentiates：This product 10ml is taken, with ether shaking extraction 2 times, each 20ml, is volatilized naturally, residue adds
Methanol 2ml makes dissolving, as test solution；Menthol reference substance separately is taken, adds methanol that solution of every 1ml containing 2mg is made, as
Reference substance solution；Tested according to thin-layered chromatography, draw 10 μ l of test solution, 5 μ l of reference substance solution, put respectively in same silica gel
On G lamellaes, with toluene-ethyl acetate（19:1）For solvent, it is unfolded, takes out, dry, spray with vanillin-sulfuric acid test solution-second
Alcohol（1:4）, clear spot is heated at 100 DEG C；In test sample chromatography, on position corresponding with reference substance chromatography, show identical
The spot of color；
3）The thin layer of subprostrate sophora differentiates：Matrine and test solution 5ml under oxymatrine item are taken, is evaporated, residue adds trichlorine
Methane 1ml makes dissolving, as test solution；Matrine reference substance separately is taken, adds chloroform that solution of every 1ml containing 1mg is made,
As reference substance solution；Tested according to thin-layered chromatography, draw 3 μ l of test solution, 2 μ l of reference substance solution, put respectively in same
On silica gel g thin-layer plate, with chloroform-methanol-strong ammonia solution（12:1:0.1）For solvent, be unfolded, take out, dry, spray with
Dilute bismuth potassium iodide test solution；In test sample chromatography, with reference substance chromatography relevant position, showing identical orange-yellow spot；
4）The discriminating of cicada slough
Mainly include amino acid in cicada slough, using liquid phase mirror method for distinguishing, 17 kinds of amino acid are screened, method is as follows：
Chromatographic condition chromatographic column：113#Kromasil100-5C18 (4.6 × 250mm, 5 μm) mobile phase：30mM sodium acetates（36%
Acetic acid tune pH to 6.50）：Acetonitrile 355：14 be mobile phase A, and 80% acetonitrile is Mobile phase B, carries out gradient elution：40 DEG C of column temperature；Stream
Fast 1.0ml/min；5 μ l of sample size；Detection wavelength 254nm；
Deriving method：Accurate 100 μ l of mixed reference substance solution, the 400 μ l of test solution of measuring adds in 10ml centrifuge tubes respectively
Enter 0.1mol/LHCl and supply 1ml, vortex mixed uniformly adds tri- second of 0.1mol/LPITC acetonitrile solutions 0.5ml, 1.0mol/L afterwards
Amine acetonitrile solution 0.5ml, vortex mixed is uniform, room temperature place 1 it is small when after, add 4ml n-hexanes, vortex mixed is uniform, room temperature
Place after ten minutes, draw lower floor's solution, to obtain the final product.
4. children according to claim 3 open the quality standard method of inspection of larynx sword spray, it is characterised in that the rock is white
The content assaying method of dish element is：According to according to liquid chromatography for measuring, specifically,
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；With methanol-water（23:77）For
Mobile phase；Detection wavelength is 275nm.
5. number of theoretical plate is calculated by Bergenin peak should be not less than 1500；
The preparation of reference substance solution takes Bergenin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made and contains Bergenin 80
The solution of μ g, to obtain the final product；
The preparation of test solution takes this product under content uniformity item, shakes up, and precision measures this product 2ml, puts in 10ml measuring bottles, adds
Methanol shakes up to scale, filters, take subsequent filtrate, to obtain the final product；
Determination method difference is accurate to draw reference substance solution and each 5 μ l of test solution, injects liquid chromatograph, measures, to obtain the final product；
This product contains bicolor per 1ml in terms of Bergenin (C14H16O9), must not be less than 0.25mg.
6. children according to claim 3 open the quality standard method of inspection of larynx sword spray, it is characterised in that the rock is bitter
Joining the content assaying method of alkali and oxymatrine is：According to high effective liquid chromatography for measuring, specifically,
Chromatographic condition and system suitability are using amino bonded silica gel as filler, with acetonitrile-phosphoric acid solution of isopropanol -3%
(85:5:10) it is mobile phase；Detection wavelength is 210nm.
7. number of theoretical plate is calculated by matrine peak should be not less than 4000；
The preparation of reference substance solution takes matrine, oxymatrine reference substance appropriate, accurately weighed, adds acetonitrile-absolute ethyl alcohol
（80:20）It is respectively prepared every 1ml 0.1mg containing matrine, the mixed solution of oxymatrine 0.075mg, to obtain the final product.
8. the preparation of test solution takes this product under content uniformity item, shake, precision measures 2ml, adds 2g neutral aluminas
（100-200 mesh）Mix thoroughly, put 70 DEG C of water bath methods, be added in neutral alumina column（100-200 mesh, 2g, internal diameter 1.5cm）On, according to
Secondary chloroform, chloroform:Methanol（7:3）Each 20ml elutions, collect eluent, put 70 DEG C of water bath methods, residue adds second
Nitrile-absolute ethyl alcohol（80:20）Dissolving, is transferred in 10ml measuring bottles, adds acetonitrile-absolute ethyl alcohol（80:20）To scale, shake, filter
Cross, take subsequent filtrate, to obtain the final product；
Determination method is accurate respectively to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures, calculates,
To obtain the final product；
This product contains subprostrate sophora per 1ml in terms of the total amount of matrine (C15H24N2O) and oxymatrine (C15H24N2O2), must not
Less than 0.15mg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711436355.5A CN107998177A (en) | 2017-12-26 | 2017-12-26 | Children open larynx sword spray and its prepare and the quality standard method of inspection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711436355.5A CN107998177A (en) | 2017-12-26 | 2017-12-26 | Children open larynx sword spray and its prepare and the quality standard method of inspection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107998177A true CN107998177A (en) | 2018-05-08 |
Family
ID=62061499
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711436355.5A Pending CN107998177A (en) | 2017-12-26 | 2017-12-26 | Children open larynx sword spray and its prepare and the quality standard method of inspection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107998177A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113655151A (en) * | 2021-09-07 | 2021-11-16 | 广东一方制药有限公司 | Animal traditional Chinese medicine standard decoction amino acid characteristic map construction and traditional Chinese medicine standard decoction and traditional Chinese medicine formula granule amino acid content detection |
| CN114081934A (en) * | 2022-01-20 | 2022-02-25 | 江西省中医药研究院 | Traditional Chinese medicine composition and quality detection method thereof |
| CN114200040A (en) * | 2021-11-25 | 2022-03-18 | 贵州三力制药股份有限公司 | Content determination method for one-test-multiple evaluation of children-type Kaihoujian spray |
| CN114965763A (en) * | 2022-05-20 | 2022-08-30 | 贵州三力制药股份有限公司 | Traditional Chinese medicine composition for oropharyngeal inflammatory diseases and multi-index quantitative determination method |
| CN115025145A (en) * | 2022-05-27 | 2022-09-09 | 贵州三力制药股份有限公司 | Method for improving pharmaceutical performance of traditional Chinese medicine composition, composition and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1686185A (en) * | 2005-03-22 | 2005-10-26 | 贵州三力制药有限责任公司 | Traditional Chinese medicina preparation for treating oral cavity, throat disease and its preparation method |
| CN103040927A (en) * | 2011-10-13 | 2013-04-17 | 贵州三力制药有限责任公司 | Open-throat sword spray used for treating throat diseases and preparation method thereof |
| CN104483404A (en) * | 2014-12-08 | 2015-04-01 | 敖云霞 | Method for measuring content of bergenin in Kaihoujian spray |
-
2017
- 2017-12-26 CN CN201711436355.5A patent/CN107998177A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1686185A (en) * | 2005-03-22 | 2005-10-26 | 贵州三力制药有限责任公司 | Traditional Chinese medicina preparation for treating oral cavity, throat disease and its preparation method |
| CN103040927A (en) * | 2011-10-13 | 2013-04-17 | 贵州三力制药有限责任公司 | Open-throat sword spray used for treating throat diseases and preparation method thereof |
| CN104483404A (en) * | 2014-12-08 | 2015-04-01 | 敖云霞 | Method for measuring content of bergenin in Kaihoujian spray |
Non-Patent Citations (5)
| Title |
|---|
| CHARGE2012: "开喉剑喷雾剂（儿童型）", 《HTTPS://WWW.DOC88.COM/P-052288833383.HTML?R=1》 * |
| 张楠等: "柱前衍生化反相高效液相色谱法测定蝉蜕中3种氨基酸含量", 《安徽中医药大学学报》 * |
| 王珍珍等: "高效液相色谱测定开喉剑喷雾剂中岩白菜素的含量", 《贵阳医学院学报》 * |
| 赵欧等: "开喉剑及组方药材山豆根、八爪金龙挥发油的GC-MS分析", 《湖北农业科学》 * |
| 赵欧等: "高效液相色谱法测定八爪金龙及组方制剂中岩白菜素的含量", 《湖北农业科学》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113655151A (en) * | 2021-09-07 | 2021-11-16 | 广东一方制药有限公司 | Animal traditional Chinese medicine standard decoction amino acid characteristic map construction and traditional Chinese medicine standard decoction and traditional Chinese medicine formula granule amino acid content detection |
| CN113655151B (en) * | 2021-09-07 | 2023-10-03 | 广东一方制药有限公司 | Construction of amino acid characteristic spectrum of animal traditional Chinese medicine standard decoction and detection of amino acid content of traditional Chinese medicine standard decoction and traditional Chinese medicine formula granule |
| CN114200040A (en) * | 2021-11-25 | 2022-03-18 | 贵州三力制药股份有限公司 | Content determination method for one-test-multiple evaluation of children-type Kaihoujian spray |
| CN114200040B (en) * | 2021-11-25 | 2023-12-08 | 贵州三力制药股份有限公司 | Content determination method for one-measurement-multiple-evaluation child type open throat sword spray |
| CN114081934A (en) * | 2022-01-20 | 2022-02-25 | 江西省中医药研究院 | Traditional Chinese medicine composition and quality detection method thereof |
| CN114965763A (en) * | 2022-05-20 | 2022-08-30 | 贵州三力制药股份有限公司 | Traditional Chinese medicine composition for oropharyngeal inflammatory diseases and multi-index quantitative determination method |
| CN114965763B (en) * | 2022-05-20 | 2023-06-16 | 贵州三力制药股份有限公司 | Traditional Chinese medicine composition for treating oral pharyngitis and multi-index quantitative determination method |
| CN115025145A (en) * | 2022-05-27 | 2022-09-09 | 贵州三力制药股份有限公司 | Method for improving pharmaceutical performance of traditional Chinese medicine composition, composition and application thereof |
| CN115025145B (en) * | 2022-05-27 | 2023-12-12 | 贵州三力制药股份有限公司 | Method and composition for improving pharmaceutical performance of traditional Chinese medicine composition and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107998177A (en) | Children open larynx sword spray and its prepare and the quality standard method of inspection | |
| CN104502518B (en) | A kind of detection method for the treatment of the Chinese medicine preparation of baby anorexia | |
| CN106198837A (en) | The quality determining method of old cough with asthma sheet | |
| CN107894488A (en) | A kind of method of quality control of concentrated type oral liquid for cough and asthma of children | |
| CN108760945B (en) | Detection method of astragalus membranaceus and astragalus membranaceus leucocyte increasing capsule | |
| CN108152437A (en) | American Ginseng reference extract and its preparation method and application | |
| CN105699500A (en) | A method of measuring contents of seven components in a Huoxiangzhengqi dripping pill through ultra-high performance liquid chromatography | |
| CN110231412A (en) | The detection method of saponin content in gynostemma pentaphylla | |
| CN109239220B (en) | A kind of quality determining method of Yupingfeng Granules | |
| CN109085285B (en) | Quality control method of changyanning granules | |
| CN110927311A (en) | Construction method of UPLC (ultra performance liquid chromatography) characteristic spectrum of dogbane leaf medicinal material and method for measuring content of flavonoid component of dogbane leaf medicinal material | |
| CN105738501A (en) | HPLC method for simultaneously determining gallic acid content and (+)-catechin content in radix sanguisorbae preparation | |
| CN108802245A (en) | A kind of root of Chinese trichosanthes or the detection method containing the drug that root of Chinese trichosanthes is raw material preparation | |
| CN107449846A (en) | The method of active ingredient in HPLC MS measure children's Anshen Bunao particles | |
| CN104678004A (en) | Quality control method for kudzuvine root and hawthorn lipid-lowering particles | |
| CN106706835A (en) | Quality detection method of trollius chinensis bunge effervescent tablets | |
| CN104849381B (en) | The method simultaneously measuring 7 kinds of Radix Astragalis based on high performance liquid chromatography-charged aerosol detectors method | |
| CN106680414A (en) | Detection method of compound ardisia japonica tablet | |
| CN110487951A (en) | A kind of quality determining method of Hedan tablet | |
| Qin et al. | Ultrasonic-assisted liquid–liquid extraction and HILIC–ELSD analysis of ginsenoside Rb1, astragaloside IV and dulcitol in sugar-free “Fufangfufangteng Heji” | |
| CN109932437A (en) | A kind of detection method of pharmaceutical composition | |
| CN113252837B (en) | Quality detection method of Jingfang mixture | |
| CN107064329B (en) | The content assaying method of five kinds of Ginsenosides in a kind of Xueshuan xinmaining Tablet | |
| CN105616946A (en) | Preparation for treating cough, preparation method and quality control method thereof | |
| CN111948331B (en) | Quality detection method of sugar-free liver-clearing granules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180508 |