CN104133003A - Establishment method of baphicacanthus cusia HPLC fingerprint - Google Patents
Establishment method of baphicacanthus cusia HPLC fingerprint Download PDFInfo
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- CN104133003A CN104133003A CN201310158540.8A CN201310158540A CN104133003A CN 104133003 A CN104133003 A CN 104133003A CN 201310158540 A CN201310158540 A CN 201310158540A CN 104133003 A CN104133003 A CN 104133003A
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- baphicacanthis cusiae
- radix baphicacanthis
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Abstract
The invention provides an establishment method of a baphicacanthus cusia HPLC fingerprint. The method includes following steps: preparing a to-be-tested sample solution, determining chromatographic conditions, and performing detection with an HPLC instrument to obtain a HPLC fingerprint of a baphicacanthus cusia medicine and extracts thereof. Overall chemical information of the baphicacanthus cusia can be comprehensively expressed and fingerprint characteristics of the baphicacanthus cusia can be determined. The fingerprint is simple in method, is good in repeatability, is many in characteristic peaks and is accurate and reliable. An established HPLC fingerprint can effectively controls quality of the baphicacanthus cusia medicine.
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
isatis indigoticafort. 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 detoxicatingly, and cool blood detumescence effect, cures mainly febrile virulent maculae, erysipelas, and influenzas etc., for the antiviral good medicine of the conventional treatment respiratory tract disease in South China and Southeast Asia, are 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 technical merit of germ plasm resource quality control has conclusive impact to Yield and quality, the market sale etc. of medicinal material.
In at present known technology, the existing quality control method of rhizoma et Radix Baphicacanthis Cusiae is mainly limited to cannot reflect chemical composition Global Information comprehensively.That traditional Chinese medicine fingerprint refers to is common in certain Chinese crude drug or Chinese patent drug, have distinctive certain class 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 can effectively make up this blank.
Therefore, the deficiency existing at present for rhizoma et Radix Baphicacanthis Cusiae, 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 its finger-print applicability inspection, the finger-print of setting 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 of setting up, degree of separation and the peak shape at the total peak of each chemical composition are good, and this finger-print is reproducible, workable, meet testing requirement, for rhizoma et Radix Baphicacanthis Cusiae medicinal material provide a kind of brand-new, 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 method obtains 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 the about 0.5g of this product powder (crossing 80 mesh sieves), accurately weighed, put in measuring bottle, precision adds 25ml aqueous solution, and weighed weight is carried out respectively ultrasonic processing, added hot reflux, extracting n-butyl alcohol 30 minutes, let cool, more weighed weight, supply respectively the weight of less loss 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;
1.2 HPLC analyze: chromatographic condition is: chromatographic column: Agilent ZorBax Eclipse XDB-C
18post (250 mm × 4.6 mm, 5 μ are m); Column temperature: 20 DEG C ~ 35 DEG C (preferably column temperature: 30 DEG C); Flow velocity: 0.8mLmin
-1~ 1.2 mLmin
-1; Chromatographic peak spectra collection scope: 190 nm～400 nm, 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%, detect wavelength 280 nm ± 2 nm, obtain rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS.
By the method for foregoing invention, by 11 batches of rhizoma et Radix Baphicacanthis Cusiae medicinal material samples being built to HPLC finger-print and analyzing relatively, 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 is in the supplementary test of embodiment 1 below, use respectively Hypersil C18 (4.6mm × 250mm, 5 μ m), Diamensil C18 (4.6mm × 250mm, 5 μ m), Agilent ZorBax Eclipse XDB-C
18(4.6mm × 250mm, 5 μ are three kinds of octadecylsilane chemically bonded silica posts m), test according to said method, and result shows, adopts Agilent C18 post to separate, and peak number is more, and degree of separation is better.In addition, above-mentioned three kinds of pillars are in the time carrying out replication, chromatographic peak (No. 5 peaks in Fig. 2) with peak area maximum calculates, number of theoretical plate reduce half (being down to 2500 from approximately 5000) can circulate mensuration number of times meter, Agilent post reaches 144 times, and Hypersil and Diamensil distinguish 75 times and 83 times, show that Agilent post is more stable significantly than the pillar of other model.Therefore it is useful adopting this post to carry out HPLC characteristic spectrum of the present invention analysis.
Inventor, in another complementary testing of embodiment 1 below, have been surprisingly found that, in the time of 30 DEG C, the significant difference in the daytime of relative retention time is than stable at other temperature.For example, in Fig. 2, at 30 DEG C, under column temperature condition, taking No. 5 peaks as basis, calculate the relative retention time approximately 1.62 at No. 10 peaks; In subsequently 10 days, 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 its RSD is 4.3%; And 25 DEG C, 35 DEG C, 40 DEG C three kinds of condition follow-on tests calculate gained RSD for 10 days and are respectively 16.8%, 15.3%, 17.7%, be presented under these column temperature conditions methodology more stable.Considering for the protection of chromatographic column and the applicability of experiment detection method, is 30 DEG C therefore select column temperature.
rhizoma et Radix Baphicacanthis Cusiae HPLC-FPS method has following significant advantage and purposes:
A. the finger-print set up can embody the overall chemical information of rhizoma et Radix Baphicacanthis Cusiae medicinal material more all sidedly;
B. this method finger-print is compared with other known rhizoma et Radix Baphicacanthis Cusiae method of quality control, and 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.
brief description of the drawings
fig. 111 crowdes of different place of production rhizoma et Radix Baphicacanthis Cusiae medicinal materials fingerprint stacking diagrams;
fig. 211 batches of different places of production 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: Agilent, 1100 high performance liquid chromatographs, DAD detecting device, Agilent Chemstation chromatographic work station, chromatographic column: Agilent ZorBax Eclipse XDB-C
18(250 mm × 4.6 mm, 5 μ are m);
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
baphicacant huscusia(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, it is pure that all the other reagent are analysis;
2 methods
The preparation of 2.1 need testing solutions: get the about 0.5g of this product powder (crossing 80 mesh sieves), accurately weighed, put in measuring bottle, precision adds 25ml aqueous solution, and weighed weight adds hot reflux 1 hour, let cool, more weighed weight, water is supplied the weight of less loss, 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;
2.2 HPLC analyze: chromatographic column: Agilent ZorBax Eclipse XDB-C
18post (250 mm × 4.6 mm, 5 μ are m); Column temperature: 30 DEG C; Flow velocity: 1.0 mLmin
-1; Chromatographic peak spectra collection scope: 190 nm～400 nm, detect wavelength 250 nm.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 and analyze relatively by being produced from 11 batches of Guizhou to rhizoma et Radix Baphicacanthis Cusiae medicinal material, 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 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 1, the preparation of need testing solution:get the about 0.5g of this product powder (crossing 80 mesh sieves), accurately weighed, put in measuring bottle, precision adds 25ml aqueous solution, and weighed weight is carried out respectively ultrasonic processing, added hot reflux, extracting n-butyl alcohol 30 minutes, let cool, more weighed weight, supply respectively the weight of less loss 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;
step 2, efficient liquid phase chromatographic analysis:the accurate need testing solution 2 μ L ~ 20 μ L sample introductions of drawing step 1, chromatographic condition is as follows: chromatographic column is Agilent ZorBax Eclipse XDB-C
18post, packing material size 2 μ m ~ 8 μ m, column length 150 mm ~ 250 mm, column temperature: 20 DEG C ~ 35 DEG C; Flow velocity: 0.8 mLmin
-1~ 1.2 mLmin
-1; Chromatographic peak spectra collection scope: 190 nm～400 nm, 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: 2% → 2% → 50% → 50%, 0.05% phosphate aqueous solution 98% → 98% → 50% → 50% (volume ratio), detects wavelength 280 nm ± 2 nm; Obtain rhizoma et Radix Baphicacanthis Cusiae HPLC finger-print;
step 3, analyze relatively:measure 11 batches of rhizoma et Radix Baphicacanthis Cusiae medicinal material sample efficient liquid-phase chromatograph finger print atlas and analyze relatively, obtaining the rhizoma et Radix Baphicacanthis Cusiae medicinal materials fingerprint that formed by its common characteristic peak, the retention time t at 10 total peaks that this finger-print has
rbe 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: retention time t
rno. 5 peak of=33.6 minutes is adenosine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105699506A (en) * | 2016-01-18 | 2016-06-22 | 吉林修正药业新药开发有限公司 | HPLC fingerprint chromatogram establishing method of Chinese patent medicine 'dibutyl particles' |
| CN109521119A (en) * | 2018-12-24 | 2019-03-26 | 广州白云山奇星药业有限公司 | A kind of measuring method of compound south isatis root granules finger-print |
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| 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 |
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| 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 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105699506A (en) * | 2016-01-18 | 2016-06-22 | 吉林修正药业新药开发有限公司 | HPLC fingerprint chromatogram establishing method of Chinese patent medicine 'dibutyl particles' |
| CN105699506B (en) * | 2016-01-18 | 2017-12-01 | 吉林修正药业新药开发有限公司 | A kind of construction method of Chinese patent drug " Erding granules " HPLC finger-prints |
| CN109521119A (en) * | 2018-12-24 | 2019-03-26 | 广州白云山奇星药业有限公司 | A kind of measuring method of compound south isatis root granules finger-print |
| 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 |
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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 |
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