CN100492000C - Method for constructing HPLC standard fingerprint pattern of semen cuscutae medicinal materials and quality identification - Google Patents
Method for constructing HPLC standard fingerprint pattern of semen cuscutae medicinal materials and quality identification Download PDFInfo
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- CN100492000C CN100492000C CNB2007100269022A CN200710026902A CN100492000C CN 100492000 C CN100492000 C CN 100492000C CN B2007100269022 A CNB2007100269022 A CN B2007100269022A CN 200710026902 A CN200710026902 A CN 200710026902A CN 100492000 C CN100492000 C CN 100492000C
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Abstract
The invention relates to construction dodder medicines HPLC standard fingerprint pattern and identification medicine quality method, used for the dodder medicine quality identification, and it discloses the powder of the trial materials ultrasonically extracting 60mins in the 20ml80% methanol solution, and filtering to obtain the trial materials solution; HPLC conditions: chromatogram column Dumas C18 column (200X4.6 mm, 5mum), column temperature 30deg.C; mobile phase acetonitrile-1% acetic acid solution, gradient elution, flow rate 1ml/min; detection wavelength 350nm. Taking 10 groups Inner Mongolia dodder medicines to determine, to find common characterized peak, with software plausible to establish a standard fingerprint pattern, there are totally six common characterized peaks, T1 to T6 peaks, retention time being 31.8min, 44.3min, 46.2min, 46.7 min, 50.1min, 68.4min; and then, compare the fingerprint pattern determined by the trial medicine and the standard fingerprint pattern, the medicine with similarity more than 0.900 is applied in the product.
Description
Technical field
The present invention relates to the construction method of seed of Chinese dodder medicinal material HPLC standard finger-print, and the method for differentiating quality of medicinal material with this standard finger-print.
Background technology
Seed of Chinese dodder medicinal material all has plantation in the many provinces of China, owing to the quality that kind, the place of production, picking time, difference caused are uneven, thereby its manufactured goods, quality drug effect and stability also are difficult to guarantee.The quality control key component of the seed of Chinese dodder is a microscopical identification in the existing state-promulgated pharmacopoeia standard.These real and fake discrimination and quality assessments for this medicinal material obviously are far from being enough.At present, more to chemical constitution study in the seed of Chinese dodder, the composition that it is reported types such as wherein containing flavones, sterol.But the onset composition of the seed of Chinese dodder also is not very clear now---be the collaborative onset of several components on earth, or one of them composition onset, also there is not ripe conclusion.Therefore to control the quality of seed of Chinese dodder medicinal material, just can not be only at two, three chemical constitutions wherein, must be controlled its material group integral body, by scientific methods this whole quality of medicinal material of comprehensively controlling of material group from forming by single chemical constitution.
That traditional Chinese medicine fingerprint is meant is common in certain Chinese crude drug (extract, Chinese patent drug), have the common spectrum of forming of distinctive composition or the collection of illustrative plates of chromatogram.It has used the notion of medical jurisprudence fingerprint identification, and the utilization modern analytical technique obtains the series of features signal of Chinese crude drug (extract, Chinese patent drug), and the collection of illustrative plates of being made up of these characteristic signals has individual uniqueness, has represented the feature of measured object.The traditional Chinese medicine fingerprint technology is differentiated the similarities and differences of Chinese crude drug (extract, Chinese patent drug) from " face " of two dimension, and what emphasize is similarity whole between collection of illustrative plates.Fingerprint pattern technology can be used in the true and false of differentiating Chinese medicine, quality of production monitoring, medicinal material to the variation of some composition of finished product, the many aspects such as mass discrepancy between the product of the same race, it provides more conventional quality standard deeper quality assessment pattern, strengthened the controllability of traditional Chinese medicine quality, be the autonomic drug quality assessment pattern of a lot of authorized by states of going abroad.Do not have under the clear and definite situation in the present stage Effective Components of Chinese Herb overwhelming majority, the traditional Chinese medicine fingerprint technology has great importance for the quality of effective control Chinese crude drug (extract, Chinese patent drug).
Current, scholar's research has been arranged detected the method for three flavones ingredients in the seed of Chinese dodder, but do not appeared in the newspapers with the disposable method that detects a plurality of compositions of the seed of Chinese dodder and set up finger-print of reversed-phase high-performance liquid chromatography with HPLC.
Summary of the invention
The purpose of this invention is to provide a kind of method that makes up seed of Chinese dodder medicinal material HPLC standard finger-print and differentiate quality of medicinal material, can be used as the thoroughly evaluating quality of medicinal material, guarantee that quality of medicinal material is stable within the specific limits, consistent, controlled.
Technical solution of the present invention is:
The first step makes up standard finger-print, and method is as follows.
(a), need testing solution preparation: seed of Chinese dodder medicinal material is worn into middle powder, takes by weighing 1.0g in tool plug conical flask, adds 20ml 80% methanol solution, and ultrasonic Extraction 60min is put coldly, filters, and gets subsequent filtrate and filters with miillpore filter (0.45 μ m), promptly.
(b), chromatographic condition: Agilent 1100 type high performance liquid chromatographs, the DAD detecting device detects wavelength 350nm; Enlightening horse C18 post (200 * 4.6mm, 5 μ m); 30 ℃ of column temperatures; Flow velocity 1ml/min; Moving phase is acetonitrile, 1% acetic acid solution, gradient elution, and the wash-out ratio is:
T(min) CH3CN(％) 1％CH3COOH(％)
0 5 95
20 5 95
80 45 55
90 5 95
(c), determination method: draw need testing solution 20ul respectively and inject hplc determination, the chromatogram of record 90min.
Measure by seed of Chinese dodder medicinal material is produced in 10 batches of Inner Mongol, find out its main common characteristic peak, through software---the HPLC standard finger-print is set up in " the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of system " match.Select 6 main common characteristic peaks altogether, be T1 to T6 peak, retention time is respectively: 31.8min, 44.3min, 46.2min, 46.7min, 50.1min, 68.4min.Though wherein T6 peak unstable (seeing the methodological study among the embodiment) still significantly detected in 24h, is the characteristic peak of the seed of Chinese dodder, so this peak is also listed in the finger-print.The relative standard deviation RSD% of described 6 peak retention times is all less than 1%.The finger-print of 10 batches of medicinal materials and the overall similarity between standard finger-print are through software---and " the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of system " calculates all greater than 0.900.
Second goes on foot, and measures the finger-print of seed of Chinese dodder medicinal material sample to be measured with above-mentioned identical method.
The 3rd step, the finger-print and the standard finger-print of seed of Chinese dodder medicinal material to be measured are compared, software---" the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004B of system " calculates overall similarity in employing, and the screening similarity is applied to production greater than 0.900 seed of Chinese dodder medicinal material.
In addition, need to prove: because fingerprint pattern technology also do not become national standard, so test and produce with seed of Chinese dodder medicinal material and all will reach respective country standard-required in the pharmacopeia among the present invention.
The present invention uses the disposable a plurality of compositions of the seed of Chinese dodder that detect of reversed-phase high-performance liquid chromatography, find out the total chromatographic peak of 10 batches of medicinal materials as characteristic peak, set up standard finger-print, just with order, ratio and the mutual restriction relation of the composition chromatographic peak that reflects in the chromatogram feature as medicinal material.Other batch medicinal materials fingerprint compares with the standard finger-print of having set up under identical chromatographic conditions, estimates quality of medicinal material by calculating overall similarity.
The present invention has the following advantages:
1. 6 principal ingredients that the principal character peak is a seed of Chinese dodder medicinal material in the standard finger-print have effectively characterized the quality of seed of Chinese dodder medicinal material, thereby realize the chemical constitution of seed of Chinese dodder maximum possible is detected, and help the monitoring in all directions to seed of Chinese dodder quality of medicinal material.
2. compare with existing method, the present invention provides new reference standard for the quality of comprehensively, accurately estimating seed of Chinese dodder medicinal material.It has promptly avoided only measuring one, two chemical constitution and the one-sidedness of judging seed of Chinese dodder total quality, and having reduced again is the artificial possibility of handling of requisite quality.
3. strengthen identification power by the present invention, and more fully estimate quality of medicinal material, guarantee that quality of medicinal material is stable within the specific limits, consistent, controlled from the angle that contained chemical constitution integral body has a common feature to the medicinal material true and false.
4. the present invention is easy, quick, stable, precision is high, good stability, be easy to grasp.
Description of drawings
Fig. 1 is a HPLC standard finger-print of the present invention.
Embodiment
One, makes up seed of Chinese dodder HPLC standard finger-print
1, instrument and reagent
1.1, instrument
Agilent 1100 liquid chromatographs, the DAD detecting device; Shanghai must the SB5200 ultrasonic cleaner.
1.2, reagent
Methyl alcohol is analyzed pure; Acetate is analyzed pure; Acetonitrile, chromatographically pure, Di Ma company produces; Ultrapure water, the Wahaha pure water; Seed of Chinese dodder medicinal material is produced in 10 batches of Inner Mongol.
Above-mentioned seed of Chinese dodder medicinal material reaches the respective country standard-required in the pharmacopeia after testing.
2, chromatographic condition
Detect wavelength 350nm; Enlightening horse C18 post (200 * 4.6mm, 5 μ m); 30 ℃ of column temperatures; Flow velocity 1ml/min; Moving phase is acetonitrile, 1% acetic acid solution, gradient elution, and the wash-out ratio is:
T(min) CH3CN(％) 1％CH3COOH(％)
0 5 95
20 5 95
80 45 55
90 5 95
3, standard finger-print is set up
3.1, need testing solution preparation: seed of Chinese dodder medicinal material is worn into middle powder, takes by weighing 1.0g in tool plug conical flask, adds the 20ml80% methanol solution, and ultrasonic Extraction 60min is put coldly, filters, and gets subsequent filtrate and filters with miillpore filter (0.45 μ m), promptly.
3.2, determination method: draw need testing solution 20ul respectively and inject hplc determination, the chromatogram of record 90min.
3.3, make up the standard fingerprint image: according to described method, by being produced seed of Chinese dodder medicinal material, 10 batches of Inner Mongol measure, find out its main common characteristic peak, through software---the HPLC standard finger-print is set up in " the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of system " match.Select 6 main common characteristic peaks altogether, be T1 to T6 peak, retention time is respectively: 31.8min, 44.3min, 46.2min, 46.7min, 50.1min, 68.4min.Though wherein T6 peak unstable (square science of law investigation) still significantly detected in 24h, is the characteristic peak of the seed of Chinese dodder, so this peak is also listed in the finger-print.The relative standard deviation RSD% of described 6 peak retention times is all less than 1%.The finger-print of 10 batches of medicinal materials and the overall similarity between standard finger-print are through software---and " the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of system " calculates all greater than 0.900.
3.4, methodological study
3.4.1, precision investigates
With same test liquid, continuous sample introduction 6 times, the precision of investigation instrument.With characteristic peak retention time and calculated by peak area, RSD%＜0.3% of 6 main common characteristic peak retention times as a result, peak area except the T6 peak changes in test liquid big, the RSD% at other peak＜3%.The result shows that the method precision is good.
3.4.2, study on the stability
With same test liquid, 0,1.5h, 3h, 4.5h, 6h, the 24h sample introduction is investigated stability of sample.Investigate the same precision of index.RSD%＜0.3% of 6 main common characteristic peak retention times as a result, peak area except the T6 peak changes in test liquid big, the RSD% at other peak＜3%.The result shows that test liquid is stable in 24h.
3.4.3, repeatability investigates
In with a collection of seed of Chinese dodder medicinal material, according to the preparation method, repeat to extract 5 parts respectively, carry out HPLC then and analyze, with the principal ingredient calculated by peak area.As a result 6 main common characteristic peak-to-peak areas except the T6 peak changes in test liquid big, other peak RSD%＜3%.The result shows that the method repeatability is good.
Two, according to described detection method and condition 2 batches of commercially available seed of Chinese dodder medicinal materials to be measured (except that 10 batches that have adopted) are measured, gathered the 90min chromatogram.This commercially available seed of Chinese dodder medicinal material to be measured after measured, the result meets seed of Chinese dodder medicinal material standard in the pharmacopeia.
The finger-print of the seed of Chinese dodder medicinal material to be measured that three, will collect and standard finger-print compare, and software---" the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004B of system " calculates overall similarity, and similarity is respectively 0.962,0.812 as a result in employing.In the two batches of medicinal materials with the similarity of standard finger-print greater than 0.900 that is a collection of, can use aborning.
Claims (1)
1, make up the method for seed of Chinese dodder medicinal material HPLC standard finger-print and discriminating quality of medicinal material, it is characterized in that:
The first step makes up standard finger-print, and method is as follows;
(a), need testing solution preparation: seed of Chinese dodder medicinal material is worn into middle powder, takes by weighing 1.0g in tool plug conical flask, adds the 20ml80% methanol solution, and ultrasonic Extraction 60min is put coldly, filters, and gets the miillpore filter filtration of subsequent filtrate with 0.45 μ m, promptly;
(b), chromatographic condition: Agilent 1100 type high performance liquid chromatographs, the DAD detecting device detects wavelength 350nm; Enlightening horse 200 * 4.6mm, the C18 post of 5 μ m; 30 ℃ of column temperatures; Flow velocity 1ml/min; Moving phase is acetonitrile, 1% acetic acid solution, gradient elution, and the wash-out ratio is: 0～20min acetonitrile is that 5%, 1% acetic acid solution is 95%; 20～80min acetonitrile changes to 45%, 1% acetic acid solution from 5% and changes to 55% from 95%; 80～90min acetonitrile changes to 5%, 1% acetic acid solution from 45% and changes to 95% from 55%;
(c), determination method: draw need testing solution 20ul respectively and inject hplc determination, the chromatogram of record 90min;
Measure by seed of Chinese dodder medicinal material is produced in 10 batches of Inner Mongol, find out its common characteristic peak, through software---" the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of system " match is set up the HPLC standard finger-print and is set up the HPLC standard finger-print; Have 6 main common characteristic peaks, be T1 to T6 peak, retention time is respectively: 31.8min, 44.3min, 46.2min, 46.7min, 50.1min, 68.4min; The relative standard deviation RSD% of described 6 peak retention times is all less than 1%; The finger-print of 10 batches of medicinal materials and the overall similarity between standard finger-print are through software---and " the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of system " calculates all greater than 0.900;
Second goes on foot, and measures the finger-print of seed of Chinese dodder medicinal material sample to be measured with above-mentioned identical method;
The 3rd step, the finger-print and the standard finger-print of seed of Chinese dodder medicinal material to be measured are compared, software---" the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004B of system " calculates overall similarity in employing, and the screening similarity is applied to production greater than 0.900 seed of Chinese dodder medicinal material.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107037146A (en) * | 2017-02-17 | 2017-08-11 | 丹东药业集团有限公司 | A kind of method of the wooden chicken granular mass control of compound |
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| CN101251518B (en) * | 2008-01-31 | 2012-05-30 | 恩施土家族苗族自治州农业科学院 | Method for establishing konjak fingerprint pattern |
| CN101975845A (en) * | 2010-09-21 | 2011-02-16 | 成都中医药大学 | Automatic detecting system for quality of traditional Chinese medicines |
| CN103616444B (en) * | 2013-11-05 | 2015-07-15 | 无锡凯夫制药有限公司 | Method for analyzing and detecting sodium aescinate for injection |
| CN104458954B (en) * | 2014-12-09 | 2016-08-24 | 广东一方制药有限公司 | A kind of dodder formulation granule finger printing and method for building up thereof |
| CN110243967B (en) * | 2019-06-26 | 2021-09-07 | 哈尔滨商业大学 | Screening method of semen cuscutae estrogen-like action quality marker and construction and application of characteristic spectrum thereof |
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| CN107037146A (en) * | 2017-02-17 | 2017-08-11 | 丹东药业集团有限公司 | A kind of method of the wooden chicken granular mass control of compound |
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