CN104483405A - Detection method of related substances in vegetable drug extract-scutellarin - Google Patents
Detection method of related substances in vegetable drug extract-scutellarin Download PDFInfo
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Abstract
The invention provides related substances in a vegetable drug extract-scutellarin and a detection method of the related substances. According to the method, an accurate detection result can be obtained, the interference caused by other substances is avoided, a double effect is achieved, an identification result is relatively objective, accurate, precise and stable.
Description
Technical field
The invention provides the detection method of the related substances in a kind of autonomic drug extract lamp-dish flower acetic.
Background technology
Often containing number of chemical composition in autonomic drug, these intrinsic chemical compositions are material bases that autonomic drug plays drug action, along with the modernization of Chinese medicine, international deeply development, in the urgent need to setting up a kind of method that Chinese medical extract is carried out to quality control and detects its complicated ingredient, finger-print arises at the historic moment.
" fingerprint " (finger printing) qualification derives from medical jurisprudence, and everyone fingerprint is had nothing in common with each other in small detail.According to these difference, by " comparison " mode, the feature differentiating everyone can be determined.Along with the development of modern biotechnology, there is DNA fingerprinting.Analyzed by DNA fingerprinting, discriminating qualification can be carried out to biosomes such as people, animal, plants.
Autonomic drug uses finger-print (especially chromatographic fingerprinting) to originate from 20 century 70s in quality control.Current traditional Chinese medicine fingerprint (fringerprinting) is used DNA fingerprinting and is developed, and is a kind of comprehensively, quantifiable chromatogram identification of means.What grow up at first is chemical composition of Chinese materia medica chromatographic fingerprinting, and if the general described finger-print in this area all refers to high performance liquid chromatography (HPLC) finger-print without special suggestion.It is the very high degree of separation utilizing HPLC to have, the chemical composition of complexity is carried out being separated and formed height different peaks form a chromatogram, the height of these chromatographic peaks and peak area represent various different chemical composition and its content respectively, and the contact of drug efficacy study result will produce Chinese medicine spectrum effect.The T.T. that the HPLC technology generally utilized at present is set up required for finger-print is longer, and degree of separation is difficult to reach comparatively ideal situation.
Autonomic drug extract Breviscapinun (principal ingredient is lamp-dish flower acetic), as the independent patent medicine of the bulk drug in pharmacopeia, is returned at 2010 editions pharmacopeia vegetable fat and extract chapters and sections.Related substances has only done the requirement of related substance peak area, but is what material on earth without bibliographical information related substances so far.And the drug effect of related substances and medicine and bad reaction have substantial connection, so chemicals all needs the contamination of labor related substance in standards of pharmacopoeia.Although Breviscapinun is used as medicine as Chinese medicine material medicine now, but its preparation is for single compound is used as medicine, especially also as injection and freeze-dried powder, such as Herba Erigerontis tablet, Breviscapini injection, Breviscapine, does not have concrete material to point out, thus also just do not have the content limit of related substances to measure, in this and external chemicals, related substance knows that discrimination and content limit measure and requires larger distance.Whether pointing out and the determination of detection method of the related substances of lamp-dish flower acetic, also had definite meaning for resolution from erigeron breviscapus.Now, the chemosynthesis of Breviscapinun also has patent CN101941999B to report, the Breviscapinun of chemosynthesis has very large difference with the Breviscapinun of extraction and isolation from erigeron breviscapus grass at related substance, thus by its with the evaluation of chemicals bulk drug very valuable and profound significance.
Summary of the invention
For the defect that prior art exists in autonomic drug extract component context of detection.Inventor, through the related substance of separation and purification Breviscapinun and a large amount of experiments, by measuring bulk drug and the Herba Erigerontis tablet of different manufacturers, obtains the detection method of related substances in lamp-dish flower acetic of the present invention.
The object of the invention is to provide a kind of related substances and detection method thereof of autonomic drug extract lamp-dish flower acetic, and can obtain testing result accurately by the method, avoid other impurity to disturb, reach effect of getting twice the result with half the effort, qualification result is more objective and accurate.
The invention provides the detection method of the related substances in a kind of autonomic drug extract lamp-dish flower acetic, this detection method comprises:
(1). the preparation of need testing solution, get test sample 20mg, add DMF 5ml and dissolve, shake up, filter, get subsequent filtrate, make the solution of test sample, the concentration of described need testing solution is that every ml is equivalent to test sample 4mg;
(2). the preparation of reference substance solution, get 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid (6-hydroxykaempferol-7-O-B-D-glucuronopyranoside, CAS NO:1258314-70-3), fleabane flower A prime (Apigenin-7-O-glucronide, CAS NO:29741-09-1), scutellarin (Scutellarein, CAS NO.:529-53-3), apiolin (Apigenin, CAS NO:520-36-5) add N, dinethylformamide makes every 1ml respectively containing 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid 0.5mg, fleabane flower A prime 0.5mg, scutellarin 0.5mg, five reference substance solution of apiolin 0.5mg,
(3). the chromatographic condition of efficient liquid phase, chromatographic column is octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.01 ~ 1%; Gradient elution program, the increasing concen-trations of mobile phase A, the descending concentrations of Mobile phase B; Flow velocity 0.1 ~ 2.0mL/min; Determined wavelength 270-350nm;
(4) mensuration of lamp-dish flower acetic related substance, draws above-mentioned test sample and each 10 μ l of reference substance solution, injects liquid chromatogram measuring instrument, measures;
(5). result detects, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, the relative retention time of itself and lamp-dish flower acetic is respectively 0.8 ~ 0.95,1.2 ~ 1.45,1.55 ~ 2.10,2.0 ~ 3.0.
Above-mentioned test sample i.e. " breviscapine B raw material medicine of extraction from plant (erigeron breviscapus) or tablet ", commercially available trade name " breviscapine active pharmaceutical ingredient " or.It is because if the lamp-dish flower acetic of synthesis, related substances is not these compositions that the application detects that protection theme is defined as " related substances in autonomic drug extract lamp-dish flower acetic " by " Herba Erigerontis tablet " the application.
In above-mentioned detection method, the chromatographic condition of described efficient liquid phase is: chromatographic column is octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.05 ~ 0.5%; Gradient elution program, the concentration of mobility A is from 30%-65%, and the concentration of Mobile phase B is from 70%-35%; Flow velocity 0.5 ~ 1.5mL/min; Determined wavelength 320-340nm; Column temperature 20-40.℃
In a preferred embodiment of the invention, detecting step is as above described in (1)-(5), and wherein, the chromatographic condition of efficient liquid phase is: chromatographic column octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 40 minutes, 55% mobility A, 45% Mobile phase B; When 50 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C; The related substances detected in the collection of illustrative plates that obtains is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, and the average relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.32,1.64,2.35.
The collection of illustrative plates obtained in above-mentioned detection method is collection of illustrative plates shown in Fig. 1.
In another preferred embodiment of the present invention, detecting step is as above described in (1)-(5), and wherein, the chromatographic condition of efficient liquid phase is: chromatographic column octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 50 minutes, 50% mobility A, 50% Mobile phase B; When 60 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C; Sample size: 10ul; The related substances detected in the collection of illustrative plates that obtains is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, and the average relative retention time of itself and lamp-dish flower acetic is respectively 0.88,1.37,1.75,2.53.
The collection of illustrative plates obtained in above-mentioned detection method is collection of illustrative plates shown in Fig. 2.
In another preferred embodiment of the present invention, detecting step is as above described in (1)-(5), and wherein, the chromatographic condition of efficient liquid phase is: chromatographic column octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 30 minutes, 50% mobility A, 50% Mobile phase B; When 40 minutes, 65% mobility A, 35% Mobile phase B; When 45 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C; Sample size: 10ul; The related substances detected in the collection of illustrative plates that obtains is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.31,1.82,1.61,2.14.
The collection of illustrative plates obtained in above-mentioned detection method is collection of illustrative plates shown in Fig. 3.
The invention provides the detection method of related substances in a kind of lamp-dish flower acetic, this detection method comprises the following steps:
(1). the preparation of need testing solution, get test sample 20mg, add DMF 5ml and dissolve, shake up, filter, get subsequent filtrate, make the solution of test sample, the concentration of described need testing solution is the solution that every ml is equivalent to that test sample 4mg makes test sample ,-
(2). get 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin, add DMF and make every 1ml respectively containing 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid 0.5mg, fleabane flower A prime 0.5mg, four reference substance solution of scutellarin 0.5mg, apiolin 0.5mg.
(3). the chromatographic condition of efficient liquid phase, chromatographic column is octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.01 ~ 1%; Gradient elution program, the increasing concen-trations of mobility A, the descending concentrations of Mobile phase B; Flow velocity 0.1 ~ 2.0mL/min; Determined wavelength 270-340nm;
Method one: mobile phase A is methyl alcohol, Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 40 minutes, 55% mobility A, 45% Mobile phase B; When 50 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.As shown in Figure 1, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin to the collection of illustrative plates obtained, apiolin, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.32,1.64,2.35.
Method two: mobile phase A is methyl alcohol, Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 50 minutes, 50% mobility A, 50% Mobile phase B; When 60 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.Sample size: 10ul; As shown in Figure 2, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin to the collection of illustrative plates obtained, apiolin, and the average relative retention time of itself and lamp-dish flower acetic is respectively 0.88,1.37,1.75,2.53.
Method three: mobile phase A is methyl alcohol, Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 30 minutes, 50% mobility A, 50% Mobile phase B; When 40 minutes, 65% mobility A, 35% Mobile phase B; When 45 minutes, 65% mobility A, 35% Mobile phase B.Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.Sample size: 10ul; As shown in Figure 3, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin to the collection of illustrative plates obtained, apiolin, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.31,1.82,1.61,2.14.
From the degree of separation at peak and quick, method three is most preferred method, and method two takes second place, and the peak interference fleabane flower A prime of lamp-dish flower acetic in method one, make the peak symmetry of fleabane flower A prime bad, small peak between is fully separated.Thus method three is better than method two, and method two is better than method one.
(4). measure: draw test sample and each 10 μ l of reference substance solution, inject liquid chromatogram measuring instrument, measure;
(5). result detects, and related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.32,1.64,2.35 in method one.Be respectively 0.88,1.37,1.75,2.53 in method two, be respectively 0.90,1.31,1.82,1.61 in method three, 2.14.
From above-mentioned detection method of the present invention, four impurity peaks that the present invention points out account for the 2.6-6.4% of relative area in breviscapine active pharmaceutical ingredient, 2.4-14.7% (method one) in Herba Erigerontis tablet; Four impurity peaks account for the 2.4-5.3% of relative area in breviscapine active pharmaceutical ingredient, 2.1-13.5% (method two) in Herba Erigerontis tablet; Four impurity peaks account for the 2.5-5.2% of relative area in breviscapine active pharmaceutical ingredient, 2.3-13.5% (method three) in Herba Erigerontis tablet; These four impurity peaks are major impurity composition in Breviscapinun raw material and tablet.In these four peaks, the bulk drug that 7 raw material pharmaceutical factories that impurity peaks 1 and impurity peaks 2 are all surveys provide and 11 medicines are looked forward to all containing in the tablet provided.The relative peak area of impurity peaks 1 in bulk drug is 1.2-4.1%, and the relative peak area in tablet is 0.9-5.4% (method one); The relative peak area of impurity peaks 1 in bulk drug is 1.1-3.2%, and the relative peak area in tablet is 0.7-4.9% (method two); The relative peak area of impurity peaks 1 in bulk drug is 1.1-3.2%, and the relative peak area in tablet is 0.8-5.0% (method three).The relative peak area of impurity peaks 2 in bulk drug is 0.8-2.3%, and the relative peak area in tablet is 1.2%-6.9% (method one); The relative peak area of impurity peaks 2 in bulk drug is 0.9-2.2%, and the relative peak area in tablet is 1.0%-6.7% (method two); The relative peak area of impurity peaks 2 in bulk drug is 0.9-2.2%, and the relative peak area in tablet is 1.1%-6.9% (method three).The relative peak area of impurity peaks 3 in bulk drug is 0-1.1%, and the relative peak area in tablet is 0.2%-1.9% (method one); The relative peak area of impurity peaks 3 in bulk drug is 0.1-0.9%, and the relative peak area in tablet is 0.2%-1.4% (method two); The relative peak area of impurity peaks 3 in bulk drug is 0.1-1.0%, and the relative peak area in tablet is 0.2%-1.5% (method three).Impurity peaks 4 almost be can't see in bulk drug, and the relative peak area in tablet is 0%-0.53% (method one); Impurity peaks 4 almost be can't see in bulk drug, and the relative peak area in tablet is 0.03%-0.5% (method two).Impurity peaks 4 almost be can't see in bulk drug, and the relative peak area in tablet is 0.07%-0.5% (method three).Therefore, if having one (except peak 3) in these four peaks or multiple peak to exist in bulk drug or tablet, especially peak 1 and peak 2 exist, and can be the raw material obtained from erigeron breviscapus grass substantially certainly, but not synthesis material.And impurity peaks 3 is owing to being that the glucuronic acid of lamp-dish flower acetic takes off the product after glucuronic acid, when having acid or highly basic to exist in separation and purification process, lamp-dish flower acetic is all easily degraded into scutellarin.Thus individualism can not illustrate whether be composite or plant extract product.
(6) with high performance liquid chromatography contrast erigeron breviscapus aerial part Aqueous extracts and four related substanceses, find that four related substanceses are the intrinsic material of erigeron breviscapus aerial part, but not catabolite or chemical reaction product.
In detection method of the present invention, the chromatographic condition of efficient liquid phase is preferred, and chromatographic column is octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.05 ~ 0.5%; Gradient elution program, the concentration of mobility A is from 30%-65%, and the concentration of Mobile phase B is from 70%-35%; Flow velocity 0.5 ~ 1.5mL/min; Determined wavelength 333-337nm; Column temperature 20-40.℃
In a preferred embodiment of the invention, the chromatographic condition of above-mentioned efficient liquid phase is preferred, and chromatographic column is octadecylsilane is filler; 250mm, adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 40 minutes, 55% mobility A, 45% Mobile phase B; When 50 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.As shown in Figure 1, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin to the collection of illustrative plates obtained, apiolin, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.32,1.64,2.35.
In another preferred embodiment of the present invention, the chromatographic condition of above-mentioned efficient liquid phase, chromatographic column is octadecylsilane is filler, 250mm, adopts gradient elution method: mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 50 minutes, 50% mobility A, 50% Mobile phase B; When 60 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.Sample size: 10ul; As shown in Figure 2, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin to the collection of illustrative plates obtained, apiolin, and the average relative retention time of itself and lamp-dish flower acetic is respectively 0.88,1.37,1.75,2.53.
In another preferred embodiment of the present invention, the chromatographic condition of above-mentioned efficient liquid phase, chromatographic column is octadecylsilane is filler, 250mm, adopts gradient elution method: mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 30 minutes, 50% mobility A, 50% Mobile phase B; When 40 minutes, 65% mobility A, 35% Mobile phase B; When 45 minutes, 65% mobility A, 35% Mobile phase B.Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.Sample size: 10ul; As shown in Figure 3, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin to the collection of illustrative plates obtained, apiolin, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.31,1.82,1.61,2.14.
Pointing out and the determination of detection method of the related substances of lamp-dish flower acetic, whether from erigeron breviscapus grass, also be there is definite meaning for resolution: because present lamp-dish flower acetic also has composite, and scutellarin is also present in [the such as leaf of the labiate height root of large-flowered skullcap (Scutellaria altissima L.), the stem of the root of large-flowered skullcap [S.baicalensis Georgi], leaf and Sculellaria barbata (S.Barbara D.Don) herb etc.] in other plant.And different related substanceses also has significantly affecting for the quality of preparation.The 6-hydroxyl Kaempferol-7-O-beta d glucopyranosiduronic acid wherein pointed out, for be separated first from erigeron breviscapus plant, was only separated be in the past inside two kinds of bachelor's-button (Centaurea).
The separation method of the related substances of lamp-dish flower acetic is by related substances in lamp-dish flower acetic raw material by the efficient liquid phase enrichment of preparative, is separated after obtaining sterling, by the structure of various method of spectroscopy authenticating compound.Then the related substances obtained and lamp-dish flower acetic raw material are carried out high-efficient liquid and compare retention time and DAD collection of illustrative plates, finally add and carry out high-efficient liquid in a small amount of pure compound to lamp-dish flower acetic material solution and compare.Only have when retention time overlaps, DAD collection of illustrative plates peak purity reaches more than 90%, just determines that this compound is related substances.
Contribution of the present invention is: by the method for efficient liquid phase, point out the related substance of the Breviscapinun of separation and purification from erigeron breviscapus, and by the method for efficient liquid phase, related substance is quantitatively detected, for in autonomic drug extract, the fingerprint map analyzing of related substances provides new thinking, the especially material that is used as medicine with single component of plant extracts.Thus established material base for studying the bad reaction relevant with impurity.
The foundation of analyzing detecting method and the investigation of influence factor.
1. the selection of chromatographic column and chromatography eluant solvent
The principle that the eluting solvent of chromatogram is selected generally will in conjunction with three indexs, polarity parameters, the dipole moment of detection compound and solubility parameter etc.When being separated the related substance of Breviscapinun, consider that the related substance of Breviscapinun is mostly the phenolic constituent of flavonoids, slant acidity, thus chromatographic column that is neutral and slant acidity is selected, (or) in eluant, eluent, add phosphoric acid or organic acid (such as formic acid or acetic acid), there is good performance to the symmetry of peak type and degree of separation.Therefore select octadecylsilane to be filler, methyl alcohol, acetonitrile, water acid adding is a kind of, and two kinds or three kinds of solvents are separated as eluant, eluent.
2. the selection of determined wavelength: under above-mentioned chromatographic condition, carries out spectral scan to need testing solution within the scope of 200 ~ 400nm, per sample 3D chromatogram, to choose the determined wavelength that can provide maximum chromatographic peak information.And under test finds single wavelength, be difficult to retain all primary fingerprint peaks of chromatographic fingerprinting of this compound.At 330 ~ 350nm place, chromatographic peak information is many, comparatively concentrated, and baseline is relatively more steady, and under this wavelength, collection of illustrative plates remains desired primary fingerprint peak.Therefore optimum chooses 335nm wavelength as determined wavelength, the wavelength of 330-340nm also can be selected as determined wavelength.
3 methodological studies:
3.1 Precision Experiments: continuous sample introduction 6 times, record each total peak relative retention time and stablize, and the RSD of each total peak relative peak area are less than 3%.Meet the relevant regulations of precision test in " technical requirement (provisional) of traditional Chinese medicine finger-print research ".
3.2 replica tests: each total peak relative retention time is stablized, the RSD of each total peak relative peak area is less than 3%, meets the relevant regulations of replica test in " technical requirement (provisional) of traditional Chinese medicine finger-print research ".
Same need testing solution is got in 3.3 stability tests, respectively at 0,4,8,12,16,20,24h sample introduction, records each total peak relative retention time and relative peak area.Test sample is stable in 24h.
Outstanding substantive distinguishing features of the present invention is, by the detection method of high performance liquid chromatography, the related substance from plant origin extraction and isolation lamp-dish flower acetic is out carried out qualitative, quantitative mensuration, thus make related substance obtain clear and definite pointing out and quantitative measurement, for taking Breviscapinun as the security of preparation of bulk drug, stability, consistance provides solid material base.And demonstrating four related substances is intrinsic materials in erigeron breviscapus plant, but not the material making a variation when extraction purification and come.Simultaneously this method of inspection can reach the effect that a survey is commented more, can under same liquid-phase condition efficiently qualitative and quantitative detection go out the related substances of four lamp-dish flower acetics, the result that the method obtains is more accurate, stable, has good consistance.Meaning of this invention is also the bulk drug be used as medicine with single component in Chinese medicine and various dosage formulation to dress, for material base has been established in the quality standard development of Chinese medicine with the quality standard of chemicals in the world.
Accompanying drawing explanation
Fig. 1 is Breviscapinun and the related substance HPLC-UV detection of method one mensuration;
Wherein, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 40 minutes, 55% mobility A, 45% Mobile phase B; When 50 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C, sample size: 10ul.
Fig. 2 is Breviscapinun and the related substance HPLC-UV detection of method two mensuration;
Wherein, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 50 minutes, 50% mobility A, 50% Mobile phase B; When 60 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.Sample size: 10ul.
Fig. 3 is Breviscapinun and the related substance HPLC-UV detection of method three mensuration;
Wherein mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 30 minutes, 50% mobility A, 50% Mobile phase B; When 40 minutes, 65% mobility A, 35% Mobile phase B; When 45 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C; Sample size: 10ul.
In Fig. 1-3, horizontal ordinate is retention time (min), and ordinate is signal intensity (mAU);
Wherein, No. 1 peak is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid; No. 2 peaks are oil lamp A primes; No. 3 peaks are scutellarins; No. 4 peaks are apiolins.
Embodiment
Describe the present invention in detail below in conjunction with drawings and Examples, but do not limit practical range of the present invention.
Embodiment one. the separation qualification of four kinds of related substances:
Get 7 breviscapine active pharmaceutical ingredients provided and 11 Herba Erigerontis tablets provided carry out high performance liquid chromatography detection, choose the separation that the higher bulk drug of its related substances carries out related substance, qualification.Get 20g breviscapine active pharmaceutical ingredient, dissolve with DMF, be separated with high performance preparative liquid chromatography, chromatographic column is Shimadzu HederaODS-P 30 × 250mm, 50ml/min, determined wavelength 335nm.With 26% methyl alcohol-0.05% aqueous formic acid for eluant, eluent, receive four related substances respectively according to ultraviolet.Separation obtains compound 1, and 2,3,4.Respectively compound 1,2,3 and 4 is done nuclear magnetic resonance map and high resolution mass spectrum.Compound 1,2,3 and 4 is accredited as through atlas analysis: 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin.
The assay method of breviscapine active pharmaceutical ingredient related substance: precision takes 20mg breviscapine active pharmaceutical ingredient, add N, dinethylformamide 5ml dissolves, shake up, filter, get subsequent filtrate, make the solution of test sample, the concentration of described need testing solution is the solution that every ml is equivalent to that test sample 4mg makes test sample.Get 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin, adds DMF and makes every 1ml respectively containing 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid 0.5mg, fleabane flower A prime 0.5mg, four reference substance solution of scutellarin 0.5mg, apiolin 0.5mg, inject high performance liquid chromatograph respectively by test sample and 4 reference substances.Chromatographic column is octadecylsilane is filler; 250mm; Mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 40 minutes, 55% mobility A, 45% Mobile phase B; When 50 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.The retention time of contrast reference substance and test sample and DAD chromatogram, when the DAD of reference substance is consistent with the retention time at peak in test sample and DAD collection of illustrative plates, determines classes of compounds in test sample and mark.
As shown in Figure 1, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin to test sample HPLC-UV detection, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.32,1.64,2.35.Four impurity peaks pointing out account for the 2.6-6.4% of relative area in breviscapine active pharmaceutical ingredient, 2.4-14.7% in Herba Erigerontis tablet, the bulk drug that 7 raw material pharmaceutical factories that impurity peaks 1 and impurity peaks 2 are all surveys provide and 11 medicines are looked forward to all containing in the tablet provided.The relative peak area of impurity peaks 1 in bulk drug is 1.2-4.1%, and the relative peak area in tablet is 0.9-5.4%; The relative peak area of impurity peaks 2 in bulk drug is 0.8-2.3%, and the relative peak area in tablet is 1.2%-6.9%; The relative peak area of impurity peaks 3 in bulk drug is 0-1.1%, and the relative peak area in tablet is 0.2%-1.9%; Impurity peaks 4 almost be can't see in bulk drug, and the relative peak area in tablet is 0%-0.53%.
Embodiment two. the separation qualification of four kinds of related substances:
Get 7 breviscapine active pharmaceutical ingredients provided and 11 Herba Erigerontis tablets provided carry out high performance liquid chromatography detection, choose the separation that the higher bulk drug of its related substances carries out related substance, qualification.Get 20g breviscapine active pharmaceutical ingredient, dissolve with DMF, be separated with high performance preparative liquid chromatography, chromatographic column is Shimadzu HederaODS-P 30 × 250mm, 50ml/min, determined wavelength 335nm.With 12% acetonitrile-0.05% aqueous formic acid for eluant, eluent, receive four related substances respectively according to ultraviolet.Separation obtains compound 1, and 2,3,4.Respectively compound 1,2,3 and 4 is done nuclear magnetic resonance map and high resolution mass spectrum.Compound 1,2,3 and 4 is accredited as through atlas analysis: 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin.
The assay method of breviscapine active pharmaceutical ingredient related substance: precision takes 20mg breviscapine active pharmaceutical ingredient, add N, dinethylformamide 5ml dissolves, shake up, filter, get subsequent filtrate, make the solution of test sample, the concentration of described need testing solution is the solution that every ml is equivalent to that test sample 4mg makes test sample.Get 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin, adds DMF and makes every 1ml respectively containing 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid 0.5mg, fleabane flower A prime 0.5mg, four reference substance solution of scutellarin 0.5mg, apiolin 0.5mg, inject high performance liquid chromatograph respectively by test sample and 4 reference substances.Chromatographic column is octadecylsilane is filler; 250mm; Mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 50 minutes, 50% mobility A, 50% Mobile phase B; When 60 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.Sample size: 10ul; The retention time of contrast reference substance and test sample and DAD chromatogram, when the DAD of reference substance is consistent with the retention time at peak in test sample and DAD collection of illustrative plates, determines classes of compounds in test sample and mark.
As shown in Figure 2, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin to test sample HPLC-UV detection, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.88,1.37,1.75,2.53.Four impurity peaks that the present invention points out account for the 2.4-5.3% of relative area in breviscapine active pharmaceutical ingredient, 2.1-13.5% in Herba Erigerontis tablet; These four impurity peaks are major impurity composition in Breviscapinun raw material and tablet.In these four peaks, the bulk drug that 7 raw material pharmaceutical factories that impurity peaks 1 and impurity peaks 2 are all surveys provide and 11 medicines are looked forward to all containing in the tablet provided.The relative peak area of impurity peaks 1 in bulk drug is 1.1-3.2%, and the relative peak area in tablet is 0.7-4.9%; The relative peak area of impurity peaks 2 in bulk drug is 0.9-2.2%, and the relative peak area in tablet is 1.0%-6.7%; The relative peak area of impurity peaks 3 in bulk drug is 0.1-0.9%, and the relative peak area in tablet is 0.2%-1.4%; Impurity peaks 4 almost be can't see in bulk drug, and the relative peak area in tablet is 0.03%-0.5%.
Embodiment three. the separation qualification of four kinds of related substances:
Get 7 breviscapine active pharmaceutical ingredients provided and 11 Herba Erigerontis tablets provided carry out high performance liquid chromatography detection, choose the separation that the higher bulk drug of its related substances carries out related substance, qualification.Get 20g breviscapine active pharmaceutical ingredient, dissolve with DMF, be separated with high performance preparative liquid chromatography, chromatographic column is Shimadzu HederaODS-P 30 × 250mm, 50ml/min, determined wavelength 335nm.With 26% methyl alcohol-0.05% aqueous formic acid for eluant, eluent, receive four related substances respectively according to ultraviolet.Again through more than half preparative high-performance liquid chromatographic purifying, be separated and obtain compound 1,2,3,4.Respectively compound 1,2,3 and 4 is done nuclear magnetic resonance map and high resolution mass spectrum.Compound 1,2,3 and 4 is accredited as through atlas analysis: 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin.
The assay method of breviscapine active pharmaceutical ingredient related substance: precision takes 20mg breviscapine active pharmaceutical ingredient, add N, dinethylformamide 5ml dissolves, shake up, filter, get subsequent filtrate, make the solution of test sample, the concentration of described need testing solution is the solution that every ml is equivalent to that test sample 4mg makes test sample.Get 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin, adds DMF and makes every 1ml respectively containing 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid 0.5mg, fleabane flower A prime 0.5mg, four reference substance solution of scutellarin 0.5mg, apiolin 0.5mg, inject high performance liquid chromatograph respectively by test sample and 4 reference substances.Chromatographic column is octadecylsilane is filler; 250mm;
Mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 30 minutes, 50% mobility A, 50% Mobile phase B; When 40 minutes, 65% mobility A, 35% Mobile phase B; When 45 minutes, 65% mobility A, 35% Mobile phase B.Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.Sample size: 10ul; The retention time of contrast reference substance and test sample and DAD chromatogram, when the DAD of reference substance is consistent with the retention time at peak in test sample and DAD collection of illustrative plates, determines classes of compounds in test sample and mark.
As shown in Figure 3, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin to test sample HPLC-UV detection, apiolin, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.31,1.82,1.61,2.14.Four impurity peaks pointing out account for the 2.5-5.2% of relative area in breviscapine active pharmaceutical ingredient, 2.3-13.5% in Herba Erigerontis tablet; These four impurity peaks are major impurity composition in Breviscapinun raw material and tablet.In these four peaks, the bulk drug that 7 raw material pharmaceutical factories that impurity peaks 1 and impurity peaks 2 are all surveys provide and 11 medicines are looked forward to all containing in the tablet provided.The relative peak area of impurity peaks 1 in bulk drug is 1.1-3.2%, and the relative peak area in tablet is 0.8-5.0%; The relative peak area of impurity peaks 2 in bulk drug is 0.9-2.2%, and the relative peak area in tablet is 1.1%-6.9%; The relative peak area of impurity peaks 3 in bulk drug is 0.1-1.0%, and the relative peak area in tablet is 0.2%-1.5%; Impurity peaks 4 almost be can't see in bulk drug, and the relative peak area in tablet is 0.07%-0.5%.
Erigeron breviscapus aerial part pure water is added hot reflux half an hour, water intaking extract filters, get subsequent filtrate and be made into test sample, get 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin, add N, dinethylformamide makes every 1ml respectively containing 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid 0.5mg, fleabane flower A prime 0.5mg, scutellarin 0.5mg, four reference substance solution of apiolin 0.5mg, test sample and 4 reference substances are injected high performance liquid chromatograph respectively and injects high performance liquid chromatograph, chromatographic column is octadecylsilane is filler, 250mm, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%, gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B, when 30 minutes, 50% mobility A, 50% Mobile phase B, when 40 minutes, 65% mobility A, 35% Mobile phase B, when 45 minutes, 65% mobility A, 35% Mobile phase B.Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C.Sample size: 100ul.The retention time of contrast reference substance and test sample and DAD chromatogram, when the DAD of reference substance is consistent with the retention time at peak in test sample and DAD collection of illustrative plates, determines classes of compounds in test sample and mark.Find that four test samples are that erigeron breviscapus aerial part is intrinsic, but not change in extraction purification process and obtain.
Claims (8)
1. the detection method of the related substances in autonomic drug extract lamp-dish flower acetic, this detection method comprises:
(1). the preparation of need testing solution, get test sample 20mg, add DMF 5ml and dissolve, shake up, filter, get subsequent filtrate, make the solution of test sample, the concentration of described need testing solution is that every ml is equivalent to test sample 4mg;
(2). the preparation of reference substance solution, get 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, fleabane flower A prime, scutellarin, apiolin adds DMF and makes every 1ml respectively containing 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid 0.5mg, fleabane flower A prime 0.5mg, four reference substance solution of scutellarin 0.5mg, apiolin 0.5mg;
(3). the chromatographic condition of efficient liquid phase, chromatographic column is octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.01 ~ 1%; Gradient elution program, the increasing concen-trations of mobile phase A, the descending concentrations of Mobile phase B; Flow velocity 0.1 ~ 2.0mL/min; Determined wavelength 270-350nm;
(4) mensuration of lamp-dish flower acetic related substance, draws above-mentioned test sample and each 10 μ l of reference substance solution, injects liquid chromatogram measuring instrument, measures;
(5). result detects, related substances is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, the relative retention time of itself and lamp-dish flower acetic is respectively 0.8 ~ 0.95,1.2 ~ 1.45,1.55 ~ 2.10,2.0 ~ 3.0.
2. detection method as claimed in claim 1, wherein, the chromatographic condition of efficient liquid phase is: chromatographic column is octadecylsilane is filler; Adopt gradient elution, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.05 ~ 0.5%; Gradient elution program, the concentration of mobility A is from 30%-65%, and the concentration of Mobile phase B is from 70%-35%; Flow velocity 0.5 ~ 1.5mL/min; Determined wavelength 320-340nm; Column temperature 20-40 DEG C.
3. detection method as claimed in claim 2, wherein, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 40 minutes, 55% mobility A, 45% Mobile phase B; When 50 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C; The related substances detected in the collection of illustrative plates that obtains is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, and the average relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.32,1.64,2.35.
4. detection method as claimed in claim 3, wherein, detecting the collection of illustrative plates obtained is Fig. 1.
5. detection method as claimed in claim 2, wherein, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 50 minutes, 50% mobility A, 50% Mobile phase B; When 60 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C; Sample size: 10ul; The related substances detected in the collection of illustrative plates that obtains is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, and the average relative retention time of itself and lamp-dish flower acetic is respectively 0.88,1.37,1.75,2.53.
6. detection method as claimed in claim 5, wherein, detecting the collection of illustrative plates obtained is Fig. 2.
7. detection method as claimed in claim 2, wherein, mobile phase A is methyl alcohol, and Mobile phase B is the phosphate aqueous solution containing 0.1%; Gradient elution program, when 0 minute, 30% mobility A, 70% Mobile phase B; When 30 minutes, 50% mobility A, 50% Mobile phase B; When 40 minutes, 65% mobility A, 35% Mobile phase B; When 45 minutes, 65% mobility A, 35% Mobile phase B; Flow velocity 1.0mL/min; Determined wavelength 335nm; Column temperature 30 DEG C; Sample size: 10ul; The related substances detected in the collection of illustrative plates that obtains is 6-hydroxyl Kaempferol-7-O-beta-glucuronic acid, oil lamp A prime, scutellarin, apiolin, and the relative retention time of itself and lamp-dish flower acetic is respectively 0.90,1.31,1.82,1.61,2.14.
8. detection method as claimed in claim 7, wherein, detecting the collection of illustrative plates obtained is Fig. 3.
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