CN104133003B - The method for building up of rhizoma et Radix Baphicacanthis Cusiae HPLC finger-print - Google Patents
The method for building up of rhizoma et Radix Baphicacanthis Cusiae HPLC finger-print Download PDFInfo
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- CN104133003B CN104133003B CN201310158540.8A CN201310158540A CN104133003B CN 104133003 B CN104133003 B CN 104133003B CN 201310158540 A CN201310158540 A CN 201310158540A CN 104133003 B CN104133003 B CN 104133003B
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Abstract
The invention provides a kind of HPLC fingerprint pattern quality control method of rhizoma et Radix Baphicacanthis Cusiae, comprise the preparation of need testing solution, the determination of chromatographic condition, high performance liquid chromatograph is used to measure, obtain rhizoma et Radix Baphicacanthis Cusiae medicinal material and extractive HPLC fingerprint thereof, have expressed the overall chemical information of rhizoma et Radix Baphicacanthis Cusiae more all sidedly, determine its fingerprint characteristic, this finger-print has that method is easy, reproducible, characteristic peak is many, feature accurately and reliably.The HPLC finger-print set up can control the quality of rhizoma et Radix Baphicacanthis Cusiae medicinal material effectively.
Description
Technical field
The present invention relates to a kind of rhizoma et Radix Baphicacanthis Cusiae medicinal material HPLC(high performance liquid chromatography) method for building up of finger-print, and the HPLC standard finger-print of the rhizoma et Radix Baphicacanthis Cusiae medicinal material of gained thus, belong to drug quality analysis technical field.
Background technology
Rhizoma et Radix Baphicacanthis Cusiae derives from acanthaceous vegetable acanthaceous indigo
baphicacanthuscusia(Nees) the dry root and rhizome of Bremek., itself and northern Radix Isatidis (cruciferae isatis
isatisindigoticafort. root) all there is difference the aspect such as Yu Jiyuan, chemical composition component, clinical practice.Rhizoma et Radix Baphicacanthis Cusiae is one of Guizhou Province's genunie medicinal materials, has clearing heat and detoxicating, and cool blood is subsided a swelling effect, and curing mainly febrile virulent maculae, erysipelas, influenza etc., is the antiviral good medicine of the treatment respiratory tract disease that South China and Southeast Asia are commonly used, and is main mainly with cultivation.But along with its market progressively expands, medicinal material demand increases, existing artificial growth rhizoma et Radix Baphicacanthis Cusiae has become the important ring in this medical supply source, therefore the Yield and quality, market sale etc. of the technical merit of germ plasm resource quality control on medicinal material have conclusive impact.
In technology known at present, the existing quality control method of rhizoma et Radix Baphicacanthis Cusiae is mainly limited to cannot reflects chemical composition Global Information comprehensively.Traditional Chinese medicine fingerprint refer to common in certain Chinese crude drug or Chinese patent drug, there is certain class distinctive or the number chromatogram of constituents or the collection of illustrative plates of spectrum.Rhizoma et Radix Baphicacanthis Cusiae not yet has the foundation of its HPLC fingerprint pattern technology of report, and general finger-print Quality Control Technology effectively can make up this blank.
Therefore, for the deficiency that rhizoma et Radix Baphicacanthis Cusiae exists at present, the present invention studies rhizoma et Radix Baphicacanthis Cusiae medicinal materials fingerprint with HPLC linear gradient elution method, select the different batches rhizoma et Radix Baphicacanthis Cusiae medicinal material of collection to complete the inspection of its finger-print applicability, the finger-print set up can embody the overall chemical information of rhizoma et Radix Baphicacanthis Cusiae medicinal material more all sidedly; In the rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS set up each chemical composition have the degree of separation at peak and peak shape good, and this finger-print is reproducible, workable, meet testing requirement, for rhizoma et Radix Baphicacanthis Cusiae medicinal material provide a kind of completely newly, be generally suitable for, convenient-to-running method of quality control.
Summary of the invention
Object of the present invention provides a kind of construction method for rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS, and the rhizoma et Radix Baphicacanthis Cusiae medicinal materials fingerprint that obtains of method thus.
Rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS construction method of the present invention is as follows:
1, the construction method of rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS:
The preparation of 1.1 need testing solutions: get this product powder (crossing 80 mesh sieves) about 0.5g, accurately weighed, put in measuring bottle, precision adds 25ml aqueous solution, weighed weight, carries out ultrasonic process respectively, adds hot reflux, extracting n-butyl alcohol 30 minutes, let cool, more weighed weight, the weight of less loss is supplied respectively with solution, shake up, centrifugal, precision measures supernatant 20mL, put in evaporating dish, fling to solvent, be settled in 5ml measuring bottle, filter, to obtain final product;
1.2HPLC analyzes: chromatographic condition is: chromatographic column: AgilentZorBaxEclipseXDB-C
18post (250mm × 4.6mm, 5 μm); Column temperature: 20 DEG C ~ 35 DEG C (preferred column temperature: 30 DEG C); Flow velocity: 0.8mLmin
-1~ 1.2mLmin
-1; Chromatographic peak spectra collection scope: 190nm ~ 400nm, mobile phase is methyl alcohol-0.05% phosphoric acid water, adopt linear gradient elution mode: 0 minute → 10 minutes → 60 minutes → 65 minutes, methyl alcohol: 2% → 2% → 50% → 50%, 0.05% phosphate aqueous solution: 98% → 98% → 50% → 50%, determined wavelength 280nm ± 2nm, obtains rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS.
By the method for foregoing invention, by building HPLC finger-print to 11 batches of rhizoma et Radix Baphicacanthis Cusiae medicinal material samples and carrying out com-parison and analysis, find out its common characteristic peak (totally 10 peaks), obtain the finger-print of rhizoma et Radix Baphicacanthis Cusiae medicinal material, the retention time (t at 22 total peaks
r) be respectively: 2.1 minutes 3.9 minutes, 5.8 minutes, 6.6 minutes, 33.6 minutes, 39.3 minutes, 44.3 minutes, 47.6 minutes, 50.1 minutes, 53.0 minutes.
Inventor, in the test supplemented of Examples below 1, uses HypersilC18 (4.6mm × 250mm, 5 μm), DiamensilC18 (4.6mm × 250mm, 5 μm), AgilentZorBaxEclipseXDB-C respectively
18(4.6mm × 250mm, 5 μm) three kinds of octadecylsilane chemically bonded silica posts, test according to said method, result shows, adopt AgilentC18 post to be separated, peak number is more, and degree of separation is better.In addition, above-mentioned three kinds of pillars are when carrying out replication, calculate with the chromatographic peak (No. 5 peaks in Fig. 2) that peak area is maximum, number of theoretical plate reduce half (being down to 2500 from about 5000) can circulate mensuration number of times meter, Agilent post reaches 144 times, and Hypersil and Diamensil distinguishes 75 times and 83 times, display Agilent post is more stable significantly than the pillar of other model.Therefore to adopt this post to carry out HPLC characteristic spectrum of the present invention analysis be useful.
Inventor, in another complementary testing of Examples below 1, have been surprisingly found that, 30 DEG C time, the significant difference in the daytime of relative retention time is than stable at other temperature.Such as in fig. 2, at 30 DEG C, under column temperature condition, based on No. 5 peaks, calculate the relative retention time about 1.62 at No. 10 peaks; In 10 subsequently day, follow-on test calculates the relative retention time at these No. 10 peaks for 3 times to inventor every day, and within 10 days as a result, result is 1.61 ± 0.07, and namely its RSD is 4.3%; And 25 DEG C, 35 DEG C, 40 DEG C three kinds of condition follow-on tests, 10 days calculating gained RSD are respectively 16.8%, 15.3%, 17.7%, under being presented at these column temperature conditions, methodology is more stable.Consider the applicability of protection for chromatographic column and experiment detection method, therefore select column temperature to be 30 DEG C.
rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS method has following significant advantage and purposes:
A. set up finger-print can embody the overall chemical information of rhizoma et Radix Baphicacanthis Cusiae medicinal material more all sidedly;
B. other rhizoma et Radix Baphicacanthis Cusiae method of quality control that this method finger-print is more known, reflection chemical composition contains much information, and chromatographic peak number is many, and degree of separation is good, and quality control level is higher;
C., compared with public technology, the need testing solution preparation process of this method is simpler and more direct, chromatographic condition is easier to realize, and experimental result reappearance is better.
Accompanying drawing explanation
fig. 111 crowdes of Different sources rhizoma et Radix Baphicacanthis Cusiae medicinal materials fingerprint stacking diagrams;
fig. 211 batches of Different sources rhizoma et Radix Baphicacanthis Cusiae medicinal materials fingerprint common patterns.
Embodiment
embodiment 1: the method for building up of rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS
1 instrument, sample, reagent and reagent
1.1 instruments: Agilent1100 high performance liquid chromatograph, DAD detecting device, AgilentChemstation chromatographic work station, chromatographic column: AgilentZorBaxEclipseXDB-C
18(250mm × 4.6mm, 5 μm);
1.2 samples: 11 batches of rhizoma et Radix Baphicacanthis Cusiae medicinal material samples, respectively from different regions in Guizhou Province, are acanthaceous vegetable acanthaceous indigo through qualification
baphicacanthuscusia(Nees) dry rhizome of Bremek and root;
1.3 reagents: adenosine (lot number: 879-200001) is purchased from Chinese pharmaceutical biological product inspection institute;
1.4 reagent: methyl alcohol is chromatographically pure, water is pure water, and it is pure that all the other reagent are analysis;
2 methods
The preparation of 2.1 need testing solutions: get this product powder (crossing 80 mesh sieves) about 0.5g, accurately weighed, put in measuring bottle, precision adds 25ml aqueous solution, weighed weight, adds hot reflux 1 hour, let cool, more weighed weight, the weight of less loss is supplied with water, shake up, centrifugal, precision measures supernatant 20mL, put in evaporating dish, fling to solvent, be settled in 5ml measuring bottle, filter, to obtain final product;
2.2HPLC analyzes: chromatographic column: AgilentZorBaxEclipseXDB-C
18post (250mm × 4.6mm, 5 μm); Column temperature: 30 DEG C; Flow velocity: 1.0mLmin
-1; Chromatographic peak spectra collection scope: 190nm ~ 400nm, determined wavelength 250nm.Methyl alcohol-0.05% phosphoric acid gradient elution, condition of gradient elution is in table 1;
table 1 condition of gradient elution
3 results:by the method for foregoing invention, build HPLC finger-print by producing rhizoma et Radix Baphicacanthis Cusiae medicinal material to 11 batches of Guizhou and carry out com-parison and analysis, finding out its common characteristic peak (totally 10 peaks), obtain the finger-print of rhizoma et Radix Baphicacanthis Cusiae medicinal material, the retention time (t at 10 total peaks
r) be respectively: 2.1 minutes 3.9 minutes, 5.8 minutes, 6.6 minutes, 33.6 minutes, 39.3 minutes, 44.3 minutes, 47.6 minutes, 50.1 minutes, 53.0 minutes.
Claims (2)
1. a method for building up for rhizoma et Radix Baphicacanthis Cusiae efficient liquid-phase chromatograph finger print atlas, is characterized in that comprising the following steps:
Step one, the preparation of need testing solution: this product powder 0.5g getting 80 mesh sieves, accurately weighed, put in measuring bottle, precision adds 25ml aqueous solution, weighed weight, carry out ultrasonic process respectively, add hot reflux, extracting n-butyl alcohol 30 minutes, to let cool, more weighed weight, supply the weight of less loss respectively with solution, shake up, centrifugal, precision measures supernatant 20mL, puts in evaporating dish, flings to solvent, be settled in 5ml measuring bottle, filter, to obtain final product;
Step 2, efficient liquid phase chromatographic analysis: the need testing solution 2 μ L ~ 20 μ L sample introduction of accurate aspiration step one, chromatographic condition is as follows: chromatographic column is AgilentZorBaxEclipseXDB-C18 post, packing material size 5 μm, column length 250mm, post footpath 4.6mm, column temperature: 30 DEG C; Flow velocity: 1.0mLmin-1; Chromatographic peak spectra collection scope: 190nm ~ 400nm, mobile phase is mixed by methyl alcohol-0.05% phosphate aqueous solution by volume, adopt linear gradient elution mode: from 0 minute → 35 minutes → 40 minutes → 53 minutes → 56 minutes → 80 minutes, methyl alcohol is with volume basis: 2% → 2% → 50% → 50%, 0.05% phosphate aqueous solution with volume basis 98% → 98% → 50% → 50%, determined wavelength 280nm ± 2nm; Obtain rhizoma et Radix Baphicacanthis Cusiae HPLC finger-print;
Step 3, com-parison and analysis: measure 11 batches of rhizoma et Radix Baphicacanthis Cusiae medicinal material sample efficient liquid-phase chromatograph finger print atlas and carry out com-parison and analysis, obtain the rhizoma et Radix Baphicacanthis Cusiae medicinal materials fingerprint be made up of its common characteristic peak, the retention time tR at 10 total peaks that this finger-print has is respectively: 2.1 minutes, 3.9 minutes, 5.8 minutes, 6.6 minutes, 33.6 minutes, 39.3 minutes, 44.3 minutes, 47.6 minutes, 50.1 minutes, 53.0 minutes.
2. method according to claim 1, is characterized in that: No. 5 peak of retention time tR=33.6 minute is adenosine.
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| CN109521119B (en) * | 2018-12-24 | 2021-07-20 | 广州白云山奇星药业有限公司 | Method for determining fingerprint spectrum of compound rhizoma et radix baphicacanthis cusiae granules |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4886666A (en) * | 1987-01-27 | 1989-12-12 | Yaguang Liu | Pharmaceutical composition for inhibiting viruses and increasing immune function (PII) |
| CN102507832A (en) * | 2011-10-26 | 2012-06-20 | 上海中医药大学 | Method for measuring high performance liquid chromatography characteristic fingerprint of isatis root and isatis root preparation |
| CN102628838A (en) * | 2012-04-26 | 2012-08-08 | 福建农林大学 | Fujian herb cusia HPLC fingerprint construction method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4886666A (en) * | 1987-01-27 | 1989-12-12 | Yaguang Liu | Pharmaceutical composition for inhibiting viruses and increasing immune function (PII) |
| CN102507832A (en) * | 2011-10-26 | 2012-06-20 | 上海中医药大学 | Method for measuring high performance liquid chromatography characteristic fingerprint of isatis root and isatis root preparation |
| CN102628838A (en) * | 2012-04-26 | 2012-08-08 | 福建农林大学 | Fujian herb cusia HPLC fingerprint construction method |
Non-Patent Citations (2)
| Title |
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| 不同产地南板蓝根药材脂溶性成分的HPLC指纹图谱研究;罗霄山等;《现代中药研究与实践》;20120630;第26卷(第6期);第64-67页 * |
| 贵州不同产地南板蓝根中腺苷的含量研究;杨丹等;《中国民族民间医药》;20120315(第5期);全文 * |
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Address after: 550025 No.4, Dongqing Road, Huaxi University Town, Huaxi District, Guiyang City, Guizhou Province Patentee after: Guizhou University of Traditional Chinese Medicine Address before: No. 50, Nanming District, Nanming District, Guiyang, Guizhou Patentee before: GUIYANG College OF TRADITIONAL CHINESE MEDICINE |