CN103245733B - A kind of ageratum dripping pill authentication method - Google Patents
A kind of ageratum dripping pill authentication method Download PDFInfo
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- CN103245733B CN103245733B CN201210024320.1A CN201210024320A CN103245733B CN 103245733 B CN103245733 B CN 103245733B CN 201210024320 A CN201210024320 A CN 201210024320A CN 103245733 B CN103245733 B CN 103245733B
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- 239000006187 pill Substances 0.000 title claims abstract description 135
- 238000000034 method Methods 0.000 title claims abstract description 73
- 240000003870 Ageratum houstonianum Species 0.000 title 1
- 241000544602 Ageratum Species 0.000 claims abstract description 137
- 238000002360 preparation method Methods 0.000 claims abstract description 55
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 138
- 230000014759 maintenance of location Effects 0.000 claims description 62
- 239000000243 solution Substances 0.000 claims description 61
- 238000012360 testing method Methods 0.000 claims description 49
- 239000013558 reference substance Substances 0.000 claims description 47
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 44
- 238000010828 elution Methods 0.000 claims description 42
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 32
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- 239000012467 final product Substances 0.000 claims description 29
- 239000000706 filtrate Substances 0.000 claims description 26
- 229960000583 acetic acid Drugs 0.000 claims description 22
- 239000000284 extract Substances 0.000 claims description 22
- 239000012362 glacial acetic acid Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 22
- 239000007853 buffer solution Substances 0.000 claims description 20
- 239000000945 filler Substances 0.000 claims description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 18
- 238000004458 analytical method Methods 0.000 claims description 16
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 16
- 239000000377 silicon dioxide Substances 0.000 claims description 16
- 241000218378 Magnolia Species 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 13
- 244000061520 Angelica archangelica Species 0.000 claims description 12
- 235000001287 Guettarda speciosa Nutrition 0.000 claims description 12
- 239000001738 pogostemon cablin oil Substances 0.000 claims description 12
- 239000001335 perilla frutescens leaf extract Substances 0.000 claims description 10
- 238000005094 computer simulation Methods 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 9
- 235000019640 taste Nutrition 0.000 claims description 9
- 244000080767 Areca catechu Species 0.000 claims description 8
- 235000006226 Areca catechu Nutrition 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 244000303040 Glycyrrhiza glabra Species 0.000 claims description 8
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 claims description 8
- 235000017443 Hedysarum boreale Nutrition 0.000 claims description 8
- 235000007858 Hedysarum occidentale Nutrition 0.000 claims description 8
- 244000197580 Poria cocos Species 0.000 claims description 8
- 235000008599 Poria cocos Nutrition 0.000 claims description 8
- 239000001947 glycyrrhiza glabra rhizome/root Substances 0.000 claims description 8
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- 241001522129 Pinellia Species 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 4
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- -1 and filter Substances 0.000 claims description 2
- 239000007888 film coating Substances 0.000 claims description 2
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- 238000002844 melting Methods 0.000 claims description 2
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- 238000010586 diagram Methods 0.000 description 4
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- 239000007791 liquid phase Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- BYTORXDZJWWIKR-UHFFFAOYSA-N Hinokiol Natural products CC(C)c1cc2CCC3C(C)(CO)C(O)CCC3(C)c2cc1O BYTORXDZJWWIKR-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- FVYXIJYOAGAUQK-UHFFFAOYSA-N honokiol Chemical compound C1=C(CC=C)C(O)=CC=C1C1=CC(CC=C)=CC=C1O FVYXIJYOAGAUQK-UHFFFAOYSA-N 0.000 description 3
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- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 description 1
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- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 240000004510 Agastache rugosa Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- GRBKWAXRYIITKG-QFMFQGICSA-N Atractylodin Chemical compound C\C=C\C#CC#C\C=C\C1=CC=CO1 GRBKWAXRYIITKG-QFMFQGICSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
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- DEMKZLAVQYISIA-UHFFFAOYSA-N Liquirtin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C2OC3=CC(O)=CC=C3C(=O)C2)C=C1 DEMKZLAVQYISIA-UHFFFAOYSA-N 0.000 description 1
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- 150000001875 compounds Chemical class 0.000 description 1
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- 229930003944 flavone Natural products 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 description 1
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 description 1
- 229940025878 hesperidin Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- OLOOJGVNMBJLLR-UHFFFAOYSA-N imperatorin Chemical compound C1=CC(=O)OC2=C1C=C1C=COC1=C2OCC=C(C)C OLOOJGVNMBJLLR-UHFFFAOYSA-N 0.000 description 1
- XKVWLLRDBHAWBL-UHFFFAOYSA-N imperatorin Natural products CC(=CCOc1c2OCCc2cc3C=CC(=O)Oc13)C XKVWLLRDBHAWBL-UHFFFAOYSA-N 0.000 description 1
- DEMKZLAVQYISIA-ZRWXNEIDSA-N liquiritin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([C@H]2OC3=CC(O)=CC=C3C(=O)C2)C=C1 DEMKZLAVQYISIA-ZRWXNEIDSA-N 0.000 description 1
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- GSZUGBAEBARHAW-UHFFFAOYSA-N sophoraflavone B Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C=2OC3=CC(O)=CC=C3C(=O)C=2)C=C1 GSZUGBAEBARHAW-UHFFFAOYSA-N 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- KWVIBDAKHDJCNY-PTRUQLRHSA-N v81k1mmx3x Chemical compound C([C@]1([C@@H](C2=C)O)CC[C@H]34)C[C@H]2C[C@H]1[C@]41CCC[C@@]3(C)CN2CCO[C@H]21 KWVIBDAKHDJCNY-PTRUQLRHSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to a kind of ageratum dripping pill authentication method, it is characterized in that, the method comprises the following steps: the preparation of step 1, standard ageratum dripping pill finger-print; The preparation of step 2, ageratum dripping pill finger-print to be measured; Step 3, compare the similarity of finger-print, both are similar shows that ageratum dripping pill to be measured is qualified.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of authentication method of ageratum dripping pill.
Background technology
Ageratum dripping pill is the exclusive kind that Tianjin Tasly Pharmaceutical Co., Ltd produces, modern Chinese herbal medicine as the research and development of sky scholar's power represents formulation, has that medicine stability is high, not easily hydrolysis oxidation, the characteristic such as rapid-action, free from extraneous odour, mouthfeel are good, easy to carry.Said preparation be comprise patchouli oil, rhizoma atractylodis, dried orange peel, the bark of official magnolia 10 taste medicinal materials composition compound preparation, and only patchouli oil wherein, the root of Dahurain angelica, the bark of official magnolia 3 taste medicinal material are qualitatively or quantitatively determined in existing detection method, the quality condition of product can not be reflected comprehensively.According to consulting pertinent literature and intra-company's data, find that in dried orange peel, in aurantiamarin, the bark of official magnolia, the composition detection wavelength such as magnolol-honokiol, root of Dahurain angelica Imperatorin-Isomperatorin is all close.Through experimental study, find the mode by converting wavelength, in product, 11 main chromatographic peaks all can reach separation requirement, therefore the invention provides a kind of authentication method of ageratum dripping pill finger-print, and the quality control for said preparation provides more perfect detection method.
For realizing this object, the present invention is by the research to ageratum dripping pill efficient liquid-phase chromatograph finger print atlas, propose a kind of better ageratum dripping pill method of quality control, compensate for the deficiency of existing Quality Control Technology, further increase the quality control level of this product, the quality control of product is improved and science more.
Summary of the invention:
technical matters to be solved
The object of the invention is to provide a kind of ageratum dripping pill finger print atlas identifying method, and the ageratum dripping pill standard finger-print that obtains of method thus.
technical scheme
The invention provides a kind of ageratum dripping pill finger print atlas identifying method, the method comprises the following steps:
The preparation of step 1, standard ageratum dripping pill finger-print;
The preparation of step 2, ageratum dripping pill finger-print to be measured;
Step 3, compare the similarity of finger-print, both are similar shows that ageratum dripping pill to be measured is qualified.
Wherein, described ageratum dripping pill is made up of rhizoma atractylodis 80-240g, dried orange peel 80-240g, bark of official magnolia 80-240g, root of Dahurain angelica 120-360g, Poria cocos 120-360g, shell of areca nut 120-360g, raw tuber of pinellia 80-240g, extract of licorice root 10-30g, patchouli oil 0.8-2.4ml, perilla leaf oil 0.4-1.2ml and appropriate amount of auxiliary materials.
Preferred ageratum pill prescription is as follows:
1) rhizoma atractylodis 160g, dried orange peel 160g, bark of official magnolia 160g, root of Dahurain angelica 240g, Poria cocos 240g, shell of areca nut 240g, raw tuber of pinellia 160g, extract of licorice root 20g, patchouli oil 1.6mL and perilla leaf oil 0.8mL is got for subsequent use;
2) ten tastes more than, get rhizoma atractylodis, dried orange peel, the bark of official magnolia, the root of Dahurain angelica respectively according to the percolation (Chinese Pharmacopoeia 2000 editions annex 1O) under liquid extract and extract item, use 60% ethanol as solvent, flood and carry out diacolation after 24 hours, collect percolate and be about 8000mL, reclaim ethanol, liquid is for subsequent use; After Poria cocos adds water boil, 80 DEG C of temperature leaching secondaries, 3 hours first times, second time 2 hours, merges warm immersion liquid, and filter, filtrate is for subsequent use; Raw half summer grade cold water soak, changes a water in every 8 hours, after bubble to the saturating heart, separately adds rhizoma zingiberis 13.5g, boiling secondary, 3 hours first times, second time 2 hours, collecting decoction, and filter, filtrate is for subsequent use; Shell of areca nut boiling 3 hours, filters, and filtrate merges with above-mentioned filter liquid and filtrate, is concentrated into paste, adds extract of licorice root, patchouli oil and perilla leaf oil, mixes.Get appropriate PEG-4000, heating makes melting, adds above-mentioned thick paste, stirs evenly, make dripping pill 1025g, film coating, to obtain final product.
Wherein, described step 1, the preparation of standard ageratum dripping pill finger-print, method is as follows:
1) preparation of need testing solution,
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process (power 200W, frequency 40kHz) 10-30 minute, preferably 15 minutes, let cool, weighed weight again, supplies the weight of less loss, shakes up with methyl alcohol, filter, get subsequent filtrate, to obtain final product.
2) preparation of reference substance solution, method is as follows:
It is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolve the solution made every 1ml and contain 140 μ g, to obtain final product.
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, and obtain chromatogram, wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, preferred ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and optimum condition is in table 1; Flow velocity is 0.1-0.4ml/min, preferred 0.2ml/min; Column temperature is 20-0 DEG C, preferably 30 DEG C; Sample size is 1-3 μ L, preferably 1 μ L; Determined wavelength is 254mn-336nm, and optimal wavelength switching condition is in table 2;
Table 1 gradient elution table
Table 2 wavelength switching condition table
According to chromatogram, find that there is 11 peaks, wherein, the chromatographic peak identical with aurantiamarin reference substance peak retention time is No. 2 peaks, the relative retention time of other 10 chromatographic peaks is respectively: 0.930,1.293,1.332,1.693,2.311,2.378,2.417,2.510,2.571,2.967, the relative retention time at each peak, within ± 3%, utilizes computer simulation Similarity Measure software 2004A version, and matching is determined to obtain ageratum dripping pill standard control finger-print.
Wherein, the preparation method of step 2 ageratum dripping pill to be measured finger-print, step is as follows;
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process (power 200W, frequency 40kHz) 10-30 minute, preferably 15 minutes, let cool, weighed weight again, supplies the weight of less loss, shakes up with methyl alcohol, filter, get subsequent filtrate, to obtain final product.Solution is injected high performance liquid chromatograph, obtains chromatogram.
Chromatographic condition is wherein as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, preferred ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and optimum condition is in table 1; Flow velocity is 0.1-0.4ml/min, preferred 0.2ml/min; Column temperature is 20-40 DEG C, preferably 30 DEG C; Sample size is 1-3 μ L, preferably 1 μ L; Determined wavelength is 254mn-336nm, and optimal wavelength switching condition is in table 2;
Table 1 gradient elution table
Table 2 wavelength switching condition table
Ageratum dripping pill finger-print step 1 and step 2 obtained compares, and similarity is more than 0.90 for qualified, and similarity is more than 0.95 for conforming to, and similarity is more than 0.98 for identical.
The present invention also provides another the ageratum dripping pill authentication method derived from above-mentioned finger-print relative method, and the method comprises the following steps:
The preparation of step 1, standard ageratum dripping pill finger-print;
The preparation of step 2, ageratum dripping pill finger-print to be measured;
Step 3, the retention time of 6 main peaks on finger-print to be compared; Wherein, described step 1, the preparation of standard ageratum dripping pill finger-print, method is as follows:
1) preparation of need testing solution,
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
2) preparation of reference substance solution, method is as follows:
It is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolve the solution made every 1ml and contain 140 μ g, to obtain final product;
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, and obtain chromatogram, wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
After com-parison and analysis is carried out to the finger-print of 10 batches of ageratum dripping pills, determine 6 main chromatographic peaks, in chromatogram, the chromatographic peak identical with aurantiamarin reference substance peak retention time is No. 2 peaks, compared with No. 2 peak retention times, the retention time at all the other 5 peaks is respectively: 0.931,1.689,2.369,2.560,2.954; With 6 peaks above-mentioned in finger-print for characteristic peak, obtain standard ageratum dripping pill finger-print;
Wherein, the preparation method of step 2 ageratum dripping pill to be measured finger-print, step is as follows;
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.Solution is injected high performance liquid chromatograph, obtains chromatogram,
Wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Wherein, on step 3, the finger-print that obtains step 1 and step 2, the retention time of 6 main peaks compares; Step is as follows; The chromatographic peak identical with reference substance peak retention time is No. 2 peaks, compared with No. 2 peak retention times, the relative retention time of other 5 chromatographic peaks is respectively 0.931 (No. 1 peak), 1.689 (No. 3 peaks), 2.369 (No. 4 peaks), 2.560 (No. 5 peaks), 2.954 (No. 6 peaks).On finger-print to be measured, on the relative retention time at 6 peaks and standard finger-print, the relative retention time at 6 peaks is that product to be tested is qualified within ± 3%.
The present invention is by the foundation of above finger-print, further establish the authentication method of ageratum dripping pill, adopt the method can accurate discrimination ageratum dripping pill, according to the comparison with standard finger-print, draw discriminating conclusion, more and standard finger-print close, product quality is better, and similarity can use computer approach to confirm, is qualified if similarity is more than 0.90, similarity is more than 0.95 for conforming to, and similarity is more than 0.98 for identical.
The preparation method of the said method particularly standard control finger-print of ageratum dripping pill is through that screening obtains, and is below screening process:
1.1 instruments and reagent
Instrument: U.S. WatersH-CLASS, Empower2 workstation.
Chromatographic column: ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um);
Reagent: methyl alcohol (chromatographically pure, Tianjin Concord Technology Co., Ltd.); Acetonitrile (chromatographically pure, German MERK company); Glacial acetic acid (analyzing pure, Tianjin Chemical Reagents Factory No.1); Water is ultrapure water.
Reference substance: aurantiamarin (lot number: 110721-201014, assay use, by 95.1% purity meter);
Magnolol
(lot number: 110729-200411, assay is used); Honokiol (lot number: 110730-201011, assay use, by 98.4% purity meter); Liquiritin (lot number: 111610-200503, assay is used); Ammonium glycyrrhetate (lot number: 110731-200615, assay is used); Imperatorin (lot number: 0826-200206, assay is used); Isomperatorin (lot number: 110827-200407, differentiate with) is Nat'l Pharmaceutical & Biological Products Control Institute to be provided; Atisine chloride Atractydin (Tianjin one side scientific & technical corporation provides)
Sample: (Tianjin Tasly Pharmaceutical Co., Ltd, totally 19 batches, concrete lot number, in table 3, is all up to the standards through Product Quality Verification Centers ageratum dripping pill.
Table 3 sample message table
The selection of 1.2 chromatographic conditions
1.2.1 the selection of determined wavelength
With reference to 2010 editions " Chinese Pharmacopoeias " and pertinent literature, summarize in ageratum dripping pill the principal ingredient adopting liquid phase to measure content in 10 taste medicinal materials, and search respectively or carry out ultraviolet full wavelength scanner, determine the maximum absorption wavelength of principal ingredient, in table 4.
Table 4 each principal ingredient absorbing wavelength situation
To sum up information, preliminary project is according to each principal ingredient peak sequence and time, and the mode adopting wavelength to switch sets up analytical approach.And the principal ingredient absorbing wavelength of about more than 60% concentrates between 280-295nm and has absorption, so adopt 294nm to investigate gradient elution chromatography (GEC) condition, until tentatively determining each principal ingredient peak sequence, after the time, chromatographic peak situation under further investigation 254nm, 336nm wavelength condition, finally to determine the setting of wavelength switching condition.
1.2.2 the selection of eluent gradient, wavelength switching condition
In conjunction with aurantiamarin, magnolol, honokiol content method data in HPLC method Simultaneously test ageratum dripping pill in intra-company's " ageratum dripping pill quality standard draft ", acetonitrile-0.5% glacial acetic acid water system is adopted to carry out the investigation of condition of gradient elution, take 294nm as determined wavelength, determine each principal ingredient peak sequence and time, then determine wavelength switching condition further.
1.2.2.1 the selection of eluent gradient
With reference to existing data and document, by the adjusting and optimizing of mobile phase ratio, determine that condition of gradient elution is in table 1; Flow velocity: 0.2ml/min; Determined wavelength: 294nm; Column temperature is 30 DEG C; Sample size: 3 μ L.
Under this chromatographic condition, typical color spectrogram is shown in Fig. 1, and as seen from the figure: about have 12 main chromatographic peaks, whole chromatogram is divided into three parts, and mainly concentrate on two time periods and go out peak, Part I concentrates between 8 ~ 13min, about has 5 chromatographic peaks; Between Part II 13 ~ 20min, chromatographic peak is all very little; Part III concentrates between 20 ~ 27min, about has 7 chromatographic peaks.
1.2.2.2 the selection of wavelength switching condition
The condition of gradient elution determined of application 1.2.2.1, investigates chromatographic peak situation under 254nm, 336nm respectively, sees Fig. 2,3.
Equally chromatogram under 254nm, 336nm is divided into three parts, what contrast three chromatograms goes out peak number and chromatographic peak size, marks off five groups of chromatographic peaks altogether, analyzes and finds: first group of chromatographic peak, between 8 ~ 10min, with maximum under 294nm wavelength; Second and third, four groups of chromatographic peaks, between 10 ~ 24min, with maximum under 254nm wavelength; 5th group of chromatographic peak then obvious under 336nm chromatographic peak area maximum.
By above analysis result, setting wavelength switching condition, in table 2.
Then condition of gradient elution and wavelength switching condition are integrated unified, tentatively determine the chromatographic condition of the fingerprint analysis method of ageratum dripping pill, the chromatogram obtained is shown in Fig. 4.
The preparation of 1.3 need testing solutions
This product of getting under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process (power 200W, frequency 40kHz) 15 minutes, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.Through preliminary reference substance comparison, determine that No. 2 peaks are aurantiamarin, No. 7 peaks are honokiol, No. 10 peaks are magnolol.
The selection of 1.4 sample sizes
Find in process of the test, the symmetry of No. 2 peak aurantiamarins is poor, symmetrical factor is only about 0.80, after magnification ratio there is acromion in chromatographic peak, then think that possible cause is that aurantiamarin chromatographic peak is wrapped up in and assorted had other peak, therefore suboptimization is again carried out to condition of gradient elution, result is through repeatedly optimizing still without improvement.When then investigating the old and new's lot number aurantiamarin reference substance solution, find that aurantiamarin reference substance chromatographic peak there are differences because concentration is different, therefore the sample size of need testing solution is reduced to 1 μ L from 3 μ L, now aurantiamarin chromatographic peak symmetrical factor is 1.06, and chromatographic peak occurs without acromion after magnification ratio, then finally determine that sample size is 1 μ L, the chromatographic condition gained chromatogram after integrating under adopting 1.2 is shown in Fig. 5.
The selection of 1.5 chromatographic columns
Chromatographic column can cause larger impact to the separation case of chromatographic column because of the difference of producer, model specification and filler, filler mode difference, therefore select other 2 kinds of conventional chromatographic columns, carries out investigation and compares.
①ACQUITYUPLC-BEHShieldRP18(2.1*100mm，1.7um)；
②AgilentZORBAXSB-C18(2.1*100mm，1.8um)；
Result shows, and chromatographic column 1. middle small peak is separated poor, sees Fig. 6; Chromatographic column 2. middle chromatographic peak has Loss, sees Fig. 7.
The determination of 1.6 liquid phase analysis conditions
To sum up analyze, finally determine that ageratum dripping pill fingerprint map analyzing liquid-phase condition is as follows:
Chromatographic column: ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase: condition of gradient elution is in table 3; Flow velocity: 0.2ml/min; Column temperature is 30 DEG C; Sample size: 1 μ L; Determined wavelength: wavelength switching condition is in table 4.
1.7 with reference to peaks selection and determine
By 1.6 lower chromatographic condition reference substances, chromatographic peak in sample finger-print is positioned, correspondence goes out 6 kinds of compositions altogether, see Fig. 8, analyze and find, 6 kinds of compositions all belong to flavones ingredient, and it is the highest with content of hesperidin, therefore determine to be that aurantiamarin reference substance is provided by Nat'l Pharmaceutical & Biological Products Control Institute, and lot number is 110721-201014 with reference to peak with aurantiamarin, assay use, by 95.1% purity meter.
The preparation of object of reference solution: it is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolves the solution made every 1ml and contain 140 μ g, to obtain final product.
The Method validation of 1.8 finger-print tests
1.8.1 replica test
Get with a collection of ageratum dripping pill that (lot number: 110803), by operating under " 1.3 " item, parallel preparation 6 parts of test samples, adopt chromatographic condition under " 1.6 " item, analyze.
With the retention time of No. 2 peak aurantiamarins and peak area for 1, calculate the relative retention time (R.Rt) of other each total fingerprint peaks and the ratio of relative peak area (R.A).The results are shown in Table 5.Result repeatability is good.
The repeated relative retention time of table 5, relative peak area tables of data
1.8.2 precision test
Get with a collection of ageratum dripping pill that (lot number: 110803), by operating under " 1.3 " item, prepare 1 part of test sample continuous sample introduction 6 times, and chromatographic condition under employing " 1.6 " item, analyzes.
With the retention time of No. 2 peak aurantiamarins and peak area for 1, calculate the relative retention time (R.Rt) of other each total fingerprint peaks and the ratio of relative peak area (R.A).The results are shown in Table 6.Result precision is good.
Table 6 precision relative retention time, relative peak area tables of data
1.8.3 stability test
Get with a collection of ageratum dripping pill (lot number: 110803), by operating under " 1.3 " item, prepare 1 part of test sample in 0,2,4,6,8,12,16,20,24H sample introduction respectively, chromatographic condition under employing " 1.6 " item, analyzes.
With the retention time of No. 2 peak aurantiamarins and peak area for 1, calculate the relative retention time (R.Rt) of other each total fingerprint peaks and the ratio of relative peak area (R.A).The results are shown in Table 7.In result 24H, sample stability is good.
Table 7 stability relative retention time, relative peak area tables of data
2.1 fingerprint spectrum method analyses
By the ageratum dripping pill test sample disposal route determined above and chromatographic condition, random selecting 10 batches of ageratum dripping pills operate by under " 1.3 " item, and chromatographic condition under employing " 1.6 " item, analyzes.
2.1.1 the foundation of reference fingerprint
According to the correlation parameter given by 10 batches of ageratum dripping pill test sample UPLC collection of illustrative plates, the main chromatographic peak that ageratum dripping pill measures gained all occurred in 32 minutes, then by the technical requirement of setting up finger-print, the record 2 times of times i.e. need testing solution chromatogram of 64 minutes, show not occur chromatographic peak in 32 minutes later, see Fig. 9.
Relatively the chromatogram of 10 batch samples find that there is 11 chromatographic peaks be each batch total, the Overlay chromatograms (see Figure 10) of 10 batch samples.Separately the relative retention time at 11 total peaks in 10 batch samples and relative peak area are analyzed, with No. 2 peak aurantiamarins for reference peak, calculate relative retention time and the peak area ratio at other 10 total peaks, then the relative retention time RSD of 10 batch samples is all within 0.1%; Unimodal area is greater than the relative peak area ratio R SD of 10% all within 5.0%, in table 8,9.
Table 810 batch sample relative retention time tables of data
Table 910 batch sample relative peak area ratio data table
2.1.2 the determination of reference fingerprint
For total peak with 11 peaks in the finger-print of above-mentioned 10 batch samples, utilize computer simulation Similarity Measure software (2004A version), matching determination ageratum dripping pill reference fingerprint, see Figure 11.
With ageratum dripping pill reference fingerprint for contrast, adopt computer simulation Similarity Measure software (2004B version), the similarity of precision, repeatability, stability test data in computing methodology checking, similarity, all more than 0.995, the results are shown in Table 10.
The similarity of table 10 precision, repeatability, stability test data
2.1.3 the source attribution analysis at total peak
By test sample preparation method under " 1.3 " item, investigate the finger-print situation of ageratum medicinal extract, ageratum medicinal extract is the middle extract not comprising patchouli oil, perilla leaf oil, extract of licorice root, chromatographic condition under employing " 1.6 " item, analyze, compare with the finger-print of ageratum dripping pill sample, result collection of illustrative plates is basically identical, chromatographic peak number and highly all indifference, then first to think in ageratum dripping pill finger-print that 11 total peaks do not derive from this 3 taste medicinal material of patchouli oil, perilla leaf oil and Radix Glycyrrhizae, see that wrinkled giant hyssop just
The finger-print comparison diagram of gas medicinal extract and ageratum dripping pill sample, Figure 12.
Again by test sample preparation method under " 1.3 " item, preparation lacks the negative sample of dried orange peel, the bark of official magnolia, rhizoma atractylodis, the root of Dahurain angelica, the tuber of pinellia, the shell of areca nut, Poria cocos respectively, by chromatographic condition under " 1.6 " item, analyzes.In conjunction with existing reference substance chromatographic peak qualitatively, determine the source ownership at 11 total peaks under existence conditions respectively, almost all concentrate on rhizoma atractylodis, dried orange peel, the root of Dahurain angelica, the bark of official magnolia 4 taste medicinal material, in table 11.
The source ownership indicator gauge at a table 1111 total peak
2.1.4 the determining fingerprint pattern of sample
By test sample preparation method under " 1.3 " item, chromatographic condition under employing " 1.6 " item, investigates the finger-print situation of ageratum dripping pill 9 batch sample, analyzes.Adopt computer simulation Similarity Measure software (2004B version) calculate similarity, result similarity all more than 0.995, in table 12.
Table 12 ageratum dripping pill keeps sample the similarity tables of data of sample
2.1.5 the standard of finger-print limits
By investigating result above, tentatively formulate the fingerprint similarity of ageratum dripping pill more than 0.95, the follow-up follow-up of quality also needing a large amount of sample, data accumulation.
2.2 characteristic spectrum methods analysts
By the ageratum dripping pill test sample disposal route determined above and chromatographic condition, random selecting 10 batches of ageratum dripping pills operate by under " 1.3 " item, and chromatographic condition under employing " 1.6 " item, analyzes.
Selection 6 main peaks wherein, as the characteristic peak of ageratum dripping pill, carry out the characteristic spectrum analysis of ageratum dripping pill, take relative retention time as foundation, carry out the data statistic analysis of Method validation project and 10 batches of on-line sample.Consistent with aurantiamarin reference substance peak retention time is No. 2 peaks, and 6 characteristic peak characteristic spectrums are shown in Figure 13.
2.2.1 the replica test of characteristic spectrum
With No. 2 peaks for reference peak, the relative retention time RSD of other 5 characteristic peaks is all below 0.1%.
The replica test tables of data of table 13 characteristic spectrum
2.2.2 the precision test of characteristic spectrum
With No. 2 peaks for reference peak, the relative retention time RSD of other 5 characteristic peaks is all below 0.1%.
The precision test tables of data of table 14 characteristic spectrum
2.2.3 the stability test of characteristic spectrum
With No. 2 peaks for reference peak, the relative retention time RSD of other 5 characteristic peaks is all below 0.2%.
The stability test tables of data of table 15 characteristic spectrum
2.2.4 characteristic peak source ownership is investigated
By test sample preparation method under " 1.3 " item, investigate the finger-print situation of ageratum medicinal extract, ageratum medicinal extract is not for comprise patchouli oil, perilla leaf oil, the middle extract of extract of licorice root, chromatographic condition under employing " 1.6 " item, analyze, compare with the finger-print of ageratum dripping pill sample, result collection of illustrative plates is basically identical, chromatographic peak number and highly all indifference, then first think that 6 characteristic peaks do not derive from patchouli oil in ageratum dripping pill finger-print, perilla leaf oil and this 3 taste medicinal material of Radix Glycyrrhizae, see the finger-print comparison diagram of ageratum medicinal extract and ageratum dripping pill sample, Figure 14.
Again by test sample preparation method under " 1.3 " item, preparation lacks the negative sample of dried orange peel, the bark of official magnolia, rhizoma atractylodis, the root of Dahurain angelica, the tuber of pinellia, the shell of areca nut, Poria cocos respectively, by chromatographic condition under " 1.6 " item, analyzes.In conjunction with existing reference substance chromatographic peak qualitatively, determine the source ownership of 6 characteristic peaks under existence conditions respectively, almost all concentrate on rhizoma atractylodis, dried orange peel, the root of Dahurain angelica, the bark of official magnolia 4 taste medicinal material, in table 16.
The source ownership indicator gauge of table 166 characteristic peak
2.2.5 the sample of characteristic spectrum is investigated
The investigation of above 10 batches of on-line sample and 9 batches of samples that keep sample is carried out to the relative retention time analysis of characteristic spectrum, as a result in 19 batch samples except No. 2 peaks with reference to 5 characteristic peak RSD except peak all within 0.5%, in table 17.Then
Table 1719 batch sample relative retention time tables of data
2.2.6 the standard of characteristic spectrum limits
According to the data of above 19 batch sample characteristic spectrums, the preliminary standard formulating characteristic spectrum limits as described below:
6 main chromatographic peaks should be presented in test sample chromatogram, the chromatographic peak identical with reference substance peak retention time is No. 2 peaks, compared with No. 2 peak retention times, the relative retention time of other 5 chromatographic peaks is respectively 0.931 (No. 1 peak), 1.689 (No. 3 peaks), 2.369 (No. 4 peaks), 2.560 (No. 5 peaks), 2.954 (No. 6 peaks).The relative retention time at each peak should within relative retention time ± 3% of regulation.
Beneficial effect of the present invention is:
The present invention is by attempting multiple different elution requirement and wavelength switching condition, finally determine wavelength and switch method carries out fingerprint map analyzing chromatographic condition to ageratum dripping pill, investigate and determine the reference fingerprint at 11 total peaks and the standard finger-print of 6 main peaks, method is easy, quick, reproducible, meets related request through Method validation.
The fingerprint analysis method of a kind of ageratum dripping pill should set up with the present invention, fingerprint map analyzing has been carried out to 9 batch samples comprising 10 years, result and reference fingerprint are compared and are calculated similarity all more than 0.995, show that the method that the present invention sets up is applicable to the requirement of the quality control of product.
The fingerprint analysis method applicability of a kind of ageratum dripping pill that the present invention sets up is strong, can reflect the quality condition of product more comprehensively.
Accompanying drawing illustrates:
Fig. 1 chromatogram, its chromatographic column is ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um), wavelength 294nm
Fig. 2 chromatogram, its chromatographic column is ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um), wavelength 254nm
Fig. 3 chromatogram, its chromatographic column is ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um), wavelength 336nm
Fig. 4 chromatogram, its chromatographic column is ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um), and wavelength switches
Fig. 5 chromatogram, its chromatographic column is ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um), sample size 1 μ L
Fig. 6 chromatogram, its chromatographic column is ACQUITYUPLC--BEHShieldRP18 (2.1*100mm, 1.7um), sample size 1 μ L
Fig. 7 chromatogram, its chromatographic column is AgilentZORBAXSB-C18 (2.1*100mm, 1.8um), sample size 1 μ L
Fig. 8 chromatogram, its chromatographic column is ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um), sample size 1 μ L
Figure 92 doubly analyzes duration chromatogram, and its chromatographic column is ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um)
Figure 101 0 batch sample compares chromatogram, and its chromatographic column is ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um)
Figure 11 ageratum dripping pill reference fingerprint fitted figure
Figure 12 ageratum medicinal extract and dripping pill sample comparison diagram
The contrast characteristic spectrum of Figure 136 characteristic peak
Figure 14 ageratum medicinal extract and dripping pill sample comparison diagram
Embodiment:
Further illustrate the present invention by the following examples, but not as limitation of the present invention.
Embodiment 1, the determining fingerprint pattern of ageratum dripping pill
Get the ageratum dripping pill that lot number is 110803
Step 1, the preparation of need testing solution
The ageratum dripping pill to be measured got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process (power 200W, frequency 40kHz) 30 minutes, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Step 2, the foundation of finger-print
Adopt high performance liquid chromatography, chromatographic condition is: chromatographic column ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and condition is in table 1; Flow velocity 0.2ml/min; Column temperature is 30 DEG C; Sample size 1 μ L; Determined wavelength is 254mn-336nm, and wavelength switching condition is in table 2; Analyze need testing solution with this understanding, obtain the finger-print of ageratum dripping pill to be measured.Embodiment 2, the determining fingerprint pattern of ageratum dripping pill
Get the ageratum dripping pill that lot number is 110803
Step 1, the preparation of need testing solution
The ageratum dripping pill to be measured got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process (power 200W, frequency 40kHz) 30 minutes, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Step 2, the foundation of finger-print
Adopt high performance liquid chromatography, chromatographic condition is: chromatographic column ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase is acetonitrile-1% glacial acetic acid buffer solution for gradient elution, and condition is in table 1; Flow velocity 0.1ml/min; Column temperature is 30 DEG C; Sample size 1 μ L; Determined wavelength is 254mn-336nm, and wavelength switching condition is in table 2; Analyze need testing solution with this understanding, obtain the finger-print of ageratum dripping pill to be measured.Embodiment 3, the determining fingerprint pattern of ageratum dripping pill
Get the ageratum dripping pill that lot number is 110306
Step 1, the preparation of need testing solution
The ageratum dripping pill to be measured got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process (power 200W, frequency 40kHz) 15 minutes, let cool, more weighed weight, supply the weight of less loss with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Step 2, the foundation of finger-print
Adopt high performance liquid chromatography, chromatographic condition is: chromatographic column ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and condition is in table 1; Flow velocity 0.2ml/min; Column temperature is 30 DEG C; Sample size 2 μ L; Determined wavelength is 254mn-336nm, and wavelength switching condition is in table 2; Analyze need testing solution with this understanding, obtain the finger-print of ageratum dripping pill to be measured.
According to method provided by the invention, the UPLC reference fingerprint set up with ageratum dripping pill is contrast, and adopt computer simulation Similarity Measure software (2004B version) to calculate, similarity is 0.997.
Embodiment 4, the determining fingerprint pattern of ageratum dripping pill
Chromatographic condition changes to:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and elution requirement is in table 1; Flow velocity is 0.4ml/min, and column temperature is 20 DEG C, and sample size is 1 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is in table 2;
Table 1 gradient elution table
Table 2 wavelength switching condition table
Embodiment 5, the determining fingerprint pattern of ageratum dripping pill
Chromatographic condition changes to:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and elution requirement is in table 1; Flow velocity is 0.3ml/min, and column temperature is 40 DEG C, and sample size is 3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is in table 2;
Table 1 gradient elution table
Table 2 wavelength switching condition table
Embodiment 6, the preparation 1 of the finger-print of standard control ageratum dripping pill
1) preparation of need testing solution,
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
2) preparation of reference substance solution, method is as follows:
It is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolve the solution made every 1ml and contain 140 μ g, to obtain final product;
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, and obtain chromatogram, wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Com-parison and analysis is carried out to the finger-print of 10 batches of ageratum dripping pills, determine 11 total peaks, in chromatogram, the chromatographic peak identical with aurantiamarin reference substance peak retention time is No. 2 peaks, the relative retention time of other 10 chromatographic peaks is respectively: 0.930,1.293,1.332,1.693,2.311,2.378,2.417,2.510,2.571,2.967, the relative retention time at each peak is within ± 3%, utilize computer simulation Similarity Measure software 2004A version, matching is determined to obtain ageratum dripping pill standard control finger-print.
Embodiment 7, the preparation 2 of the finger-print of standard control ageratum dripping pill
1) preparation of need testing solution,
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process (power 200W, frequency 40kHz) 10 ~ 30 minutes, preferably 15 minutes, let cool, weighed weight again, supplies the weight of less loss, shakes up with methyl alcohol, filter, get subsequent filtrate, to obtain final product.
2) preparation of reference substance solution, method is as follows:
It is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolve the solution made every 1ml and contain 140 μ g, to obtain final product.
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, and obtain chromatogram, wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler ACQUITYUPLC-HSST3 (2.1*100mm, 1.7um); Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and elution requirement is in table 1; Flow velocity is 0.2ml/min; Column temperature is 30 DEG C; Sample size is 1 μ L; Determined wavelength is 254mn-336nm, and wavelength switching condition is in table 2;
Table 1 gradient elution table
Table 2 wavelength switching condition table
After com-parison and analysis is carried out to the finger-print of 10 batches of ageratum dripping pills, determine 6 main chromatographic peaks, the chromatographic peak identical with aurantiamarin reference substance peak retention time is No. 2 peaks, compared with No. 2 peak retention times, the retention time at all the other 5 peaks is respectively: 0.931,1.689,2.369,2.560,2.954, the relative retention time at each peak is within ± 3%, and these total peaks constitute the fingerprint characteristic of ageratum dripping pill, can be used as the standard control finger-print of ageratum dripping pill.
Embodiment 8
Step 1, the preparation of standard ageratum dripping pill finger-print, method is as follows:
1) preparation of need testing solution,
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
2) preparation of reference substance solution, method is as follows:
It is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolve the solution made every 1ml and contain 140 μ g, to obtain final product;
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, and obtain chromatogram, wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Com-parison and analysis is carried out to the finger-print of 10 batches of ageratum dripping pills, determine 11 total peaks, in chromatogram, the chromatographic peak identical with aurantiamarin reference substance peak retention time is No. 2 peaks, the relative retention time of other 10 chromatographic peaks is respectively: 0.930,1.293,1.332,1.693,2.311,2.378,2.417,2.510,2.571,2.967, the relative retention time at each peak is within ± 3%, utilize computer simulation Similarity Measure software 2004A version, matching is determined to obtain ageratum dripping pill standard control finger-print.
The preparation method of step 2 ageratum dripping pill to be measured finger-print, step is as follows;
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10 ~ 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product.Solution is injected high performance liquid chromatograph, obtains chromatogram,
Wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Step 3, ageratum dripping pill finger-print step 1 and step 2 obtained compares, and similarity is more than 0.90 is qualified.
Claims (10)
1. an ageratum dripping pill authentication method, is characterized in that, the method comprises the following steps:
The preparation of step 1, standard ageratum dripping pill finger-print;
The preparation of step 2, ageratum dripping pill finger-print to be measured;
Step 3, the similarity of finger-print to be compared;
Wherein, described step 1, the preparation of standard ageratum dripping pill finger-print, method is as follows:
1) preparation of need testing solution,
2) preparation of reference substance solution,
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, obtain chromatogram,
Wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Condition of gradient elution is as follows:
2. authentication method according to claim 1, it is characterized in that, wherein, described ageratum dripping pill is made up of rhizoma atractylodis 80-240g, dried orange peel 80-240g, bark of official magnolia 80-240g, root of Dahurain angelica 120-360g, Poria cocos 120-360g, shell of areca nut 120-360g, raw tuber of pinellia 80-240g, extract of licorice root 10-30g, patchouli oil 0.8-2.4ml, perilla leaf oil 0.4-1.2ml and appropriate amount of auxiliary materials.
3. authentication method according to claim 1, is characterized in that, wherein, described ageratum dripping pill is prepared from by the following method:
1) rhizoma atractylodis 160g, dried orange peel 160g, bark of official magnolia 160g, root of Dahurain angelica 240g, Poria cocos 240g, shell of areca nut 240g, raw tuber of pinellia 160g, extract of licorice root 20g, patchouli oil 1.6mL and perilla leaf oil 0.8mL is got for subsequent use;
2) ten tastes more than, get rhizoma atractylodis, dried orange peel, the bark of official magnolia, the root of Dahurain angelica respectively according to the percolation under liquid extract and extract item, use 60% ethanol as solvent, flood and carry out diacolation after 24 hours, collect percolate and are about 8000mL, reclaim ethanol, liquid is for subsequent use; After Poria cocos adds water boil, 80 DEG C of temperature leaching secondaries, 3 hours first times, second time 2 hours, merges warm immersion liquid, and filter, filtrate is for subsequent use; Raw half summer grade cold water soak, changes a water in every 8 hours, after bubble to the saturating heart, separately adds rhizoma zingiberis 13.5g, boiling secondary, 3 hours first times, second time 2 hours, collecting decoction, and filter, filtrate is for subsequent use; Shell of areca nut boiling 3 hours, filters, and filtrate merges with above-mentioned filter liquid and filtrate, is concentrated into paste, adds extract of licorice root, patchouli oil and perilla leaf oil, mixes; Get appropriate PEG-4000, heating makes melting, adds above-mentioned thick paste, stirs evenly, make dripping pill 1025g, film coating, to obtain final product.
4. authentication method according to claim 1, is characterized in that,
Wherein, described step 1, the preparation of standard ageratum dripping pill finger-print, method is as follows:
1) preparation of need testing solution,
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
2) preparation of reference substance solution, method is as follows:
It is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolve the solution made every 1ml and contain 140 μ g, to obtain final product;
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, obtain chromatogram,
Wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Condition of gradient elution is as follows:
Com-parison and analysis is carried out to the finger-print of 10 batches of ageratum dripping pills, determine 11 total peaks, in chromatogram, the chromatographic peak identical with aurantiamarin reference substance peak retention time is No. 2 peaks, the relative retention time of other 10 chromatographic peaks is respectively: 0.902 ~ 0.958, 1.254 ~ 1.332, 1.292 ~ 1.372, 1.642 ~ 1.744, 2.242 ~ 2.380, 2.307 ~ 2.449, 2.344 ~ 2.490, 2.435 ~ 2.585, 2.494 ~ 2.648, 2.878 ~ 3.056, utilize computer simulation Similarity Measure software 2004A version, matching is determined to obtain ageratum dripping pill standard control finger-print.
5. authentication method according to claim 1, is characterized in that,
Wherein, the preparation method of step 2 ageratum dripping pill to be measured finger-print, step is as follows;
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product; Solution is injected high performance liquid chromatograph, obtains chromatogram,
Wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Condition of gradient elution is as follows:
6. authentication method according to claim 1, is characterized in that,
Described step 3, comparing the similarity of finger-print, is that ageratum dripping pill finger-print step 1 and step 2 obtained compares, and similarity is more than 0.90 for qualified.
7. authentication method according to claim 1, is characterized in that,
Described step 3, comparing the similarity of finger-print, is that ageratum dripping pill finger-print step 1 and step 2 obtained compares, and similarity is more than 0.95 for qualified.
8. authentication method according to claim 1, is characterized in that,
Described step 3, comparing the similarity of finger-print, is that ageratum dripping pill finger-print step 1 and step 2 obtained compares, and similarity is more than 0.98 for qualified.
9. authentication method according to claim 1, is characterized in that, step is as follows:
Step 1, the preparation of the finger-print of standard control ageratum dripping pill
1) preparation of need testing solution,
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
2) preparation of reference substance solution, method is as follows:
It is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolve the solution made every 1ml and contain 140 μ g, to obtain final product;
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, obtain chromatogram,
Wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler ACQUITYUPLC-HSST3, and wherein, chromatographic column specification is 2.1*100mm, particle diameter 1.7 μm; Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and elution requirement is in table 1; Flow velocity is 0.2ml/min; Column temperature is 30 DEG C; Sample size is 1 μ L; Determined wavelength is 254mn-336nm, and wavelength switching condition is in table 2;
Table 1 gradient elution table
Table 2 wavelength switching condition table
Com-parison and analysis is carried out to the finger-print of 10 batches of ageratum dripping pills, determine 11 total peaks, in chromatogram, the chromatographic peak identical with aurantiamarin reference substance peak retention time is No. 2 peaks, the relative retention time of other 10 chromatographic peaks is respectively: 0.902 ~ 0.958, 1.254 ~ 1.332, 1.292 ~ 1.372, 1.642 ~ 1.744, 2.242 ~ 2.380, 2.307 ~ 2.449, 2.344 ~ 2.490, 2.435 ~ 2.585, 2.494 ~ 2.648, 2.878 ~ 3.056, utilize computer simulation Similarity Measure software 2004A version, matching is determined to obtain ageratum dripping pill standard control finger-print,
Step 2, the determining fingerprint pattern of ageratum dripping pill to be measured
The preparation of need testing solution
The ageratum dripping pill to be measured got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
Adopt high performance liquid chromatography, chromatographic condition is: chromatographic column ACQUITYUPLC-HSST3, and wherein, chromatographic column specification is 2.1*100mm, particle diameter 1.7 μm; Mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and condition is in table 1; Flow velocity 0.2ml/min; Column temperature is 30 DEG C; Sample size 1 μ L; Determined wavelength is 254mn-336nm, and wavelength switching condition is in table 2; Obtain the finger-print of ageratum dripping pill to be measured with this understanding;
Step 3, ageratum dripping pill finger-print step 1 and step 2 obtained compares, and similarity is more than 0.90 is qualified.
10. an ageratum dripping pill authentication method, is characterized in that, the method comprises the following steps:
The preparation of step 1, standard ageratum dripping pill finger-print;
The preparation of step 2, ageratum dripping pill finger-print to be measured;
Step 3, the retention time of 6 main peaks on finger-print to be compared;
Wherein, described step 1, the preparation of standard ageratum dripping pill finger-print, method is as follows:
1) preparation of need testing solution,
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product;
2) preparation of reference substance solution, method is as follows:
It is appropriate that precision takes aurantiamarin reference substance, adds methyl alcohol and dissolve the solution made every 1ml and contain 140 μ g, to obtain final product;
3) determination of finger-print,
Need testing solution and reference substance solution are injected high performance liquid chromatograph, obtain chromatogram,
Wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Condition of gradient elution is as follows:
After com-parison and analysis is carried out to the finger-print of 10 batches of ageratum dripping pills, determine 6 main chromatographic peaks, in chromatogram, the chromatographic peak identical with aurantiamarin reference substance peak retention time is No. 2 peaks, compared with No. 2 peak retention times, the retention time at all the other 5 peaks is respectively: 0.931,1.689,2.369,2.560,2.954;
Wherein, the preparation method of step 2 ageratum dripping pill to be measured finger-print, step is as follows;
The ageratum dripping pill got under content uniformity item is appropriate, crushes dressing, gets about 1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic process 10-30 minute, let cool, more weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter, get subsequent filtrate, to obtain final product; Solution is injected high performance liquid chromatograph, obtains chromatogram,
Wherein the chromatographic condition of high performance liquid chromatography is as follows:
Chromatographic column fixed phase take octadecylsilane chemically bonded silica as filler, and mobile phase is acetonitrile-0.5% glacial acetic acid buffer solution for gradient elution, and flow velocity is 0.1-0.4ml/min, column temperature is 20-40 DEG C, sample size is 1-3 μ L, and determined wavelength is 254mn-336nm, and wavelength switching condition is as follows:
Condition of gradient elution is as follows:
Wherein, step 3, the retention time of 6 main peaks on two finger-prints is compared; Step is as follows; The chromatographic peak identical with reference substance peak retention time is No. 2 peaks, compared with No. 2 peak retention times, the relative retention time of other 5 chromatographic peaks is respectively 0.931,1.689,2.369,2.560,2.954, and on finger-print more to be measured, on the relative retention time at 6 peaks and standard finger-print, the relative retention time at 6 peaks is that product to be tested is qualified within ± 3%.
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| CN108627588B (en) * | 2018-08-14 | 2024-03-12 | 上海和黄药业有限公司 | Detection method of Zhengqi tablet fingerprint and application thereof |
| CN109298089A (en) * | 2018-10-19 | 2019-02-01 | 南京中医药大学 | The method of quality control of rhizoma zingiberis |
| CN109342609B (en) * | 2018-12-21 | 2021-04-20 | 国药控股星鲨制药（厦门）有限公司 | Method for identifying chemical components of traditional Chinese medicine fluid extract of compound three-dimensional calcium D-pantothenate syrup |
| CN112213416B (en) * | 2020-09-03 | 2022-09-20 | 广东一方制药有限公司 | Finger print construction method and identification method of two traditional Chinese medicine formula granules |
| CN114636779B (en) * | 2022-03-29 | 2024-05-24 | 陕西盘龙药业集团股份有限公司 | Construction method of three-conversion soup reference sample freeze-dried powder fingerprint and fingerprint thereof |
| CN116183739A (en) * | 2022-11-25 | 2023-05-30 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Determination method for analyzing fingerprint of Huoxiang Zhengqi soft capsule |
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