CN105738547A - Calculus eliminating tablet fingerprint establishing method - Google Patents
Calculus eliminating tablet fingerprint establishing method Download PDFInfo
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- CN105738547A CN105738547A CN201511022764.1A CN201511022764A CN105738547A CN 105738547 A CN105738547 A CN 105738547A CN 201511022764 A CN201511022764 A CN 201511022764A CN 105738547 A CN105738547 A CN 105738547A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention provides a calculus eliminating tablet fingerprint establishing method. The method comprises the following steps: preparing a reference substance, preparing a test sample, and using high performance liquid chromatography (HPLC) to obtain the fingerprint of a calculus eliminating tablet; wherein in the chromatography and system applicability tests, octadecyl silane bonded silica gel is taken as the filling agent, acetonitrile is taken as the mobile phase (A), a phosphoric acid water solution (0.2%) is taken as the mobile phase (B), methanol is taken as the mobile phase (C), the detection wavelength is 272, and the theoretical plate number calculated by schaftoside is not less than 3000.
Description
Technical field
The present invention relates to drug measurement techniques field, be specifically related to the method for building up of a kind of JIESHITONG PIAN finger printing.
Background technology
Chinese medicine compound controls to be one of key issue restricting modernization of Chinese medicine development.Theory of Chinese medical science emphasizes the group effect of Chinese medicine, payes attention to the synergism in drug effect, in Chinese medicine one, two effective ingredient as qualitative, quantitative target far from the quality efficiently controlling and evaluating Chinese medicine, it is more difficult to reflect its safety and effectiveness.Chinese medicine compound is the compound preparation that the two above Chinese medicine composition of taste are applied to disease treatment, and the difficulty of its quality control relatively single medicinal material is more very.In recent years, Chinese medicine fingerprint and characteristic fingerprint pattern are widely used to quality control, wherein Chinese medicine characteristic spectrum means and chooses the strong chromatographic peak of some specificities or chromatographic peak assemblage characteristic finger printing from Chinese medicine fingerprint, having that it's too late Chinese medicine quality is monitored in change by characteristic fingerprint peak.
JIESHITONG PIAN is made up of multi-flavor medical materials such as Herba Desmodii Styracifolii, Folium Pyrrosiae, Herba Plantaginis, Poria, has effect of clearing away heat and promoting diuresis, Tonglin Paishi, pain easing and hemostasis.Clinic is used for treatment urinary system infection, cystitis, oedema due to nephritis, lithangiuria, hematuria, drenches drop muddy, and urethra causalgia etc. is recorded in the Sanitation Ministry medicine standard Traditional Chinese medicine historical preparation the 13rd.
The method of quality control Main Basis standard WS3-B-2589-97 quality standard of current JIESHITONG PIAN only has character and check item, it is difficult to control the quality of medicine.
Summary of the invention
The present invention is directed to existing JIESHITONG PIAN quality standard relatively backward, only have character, check project, the problem that can not effectively control patent medicine quality, the method for building up of a kind of JIESHITONG PIAN finger printing is provided, by the foundation of JIESHITONG PIAN fingerprint spectrum method, and the standard finger-print of the thus JIESHITONG PIAN that method obtains, efficiently control the quality of JIESHITONG PIAN, make up the deficiency of existing Quality Control Technology, so that it is guaranteed that the effectiveness of JIESHITONG PIAN, safety and stability.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The method for building up of a kind of JIESHITONG PIAN finger printing, the method includes adopting high performance liquid chromatography that JIESHITONG PIAN is detected, and wherein chromatographic condition includes；
Chromatographic column: with octadecylsilane chemically bonded silica for filler；
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.2% phosphate aqueous solution, and mobile phase C is methanol；
Adopting gradient elution, elution program is as follows, and wherein mobile phase ratio is volume basis specific volume:
0～10min, mobile phase A is 10%, and Mobile phase B is 85%；Mobile phase C is 5%；
10～20min, mobile phase A is 10%；Mobile phase B is 85% → 82%；Mobile phase C is 5% → 8%；
20～30min, mobile phase A is 10% → 12%；Mobile phase B is 82% → 80%；Mobile phase C is 8%；
30～60min, mobile phase A is 12% → 17%；Mobile phase B is 80% → 75%；Mobile phase C is 8%；
Detection wavelength is 272；
Theoretical cam curve calculates by Schaftoside and is not less than 3000.
The method for building up of described JIESHITONG PIAN finger printing, also includes preparing reference substance solution by following steps: take Schaftoside reference substance, accurately weighed, adds methanol and makes every ml solution containing Schaftoside 0.05mg, to obtain final product.
The method for building up of described JIESHITONG PIAN finger printing, also includes preparing need testing solution by following steps: JIESHITONG PIAN powder 1g, adds 60% methanol 30ml, reflux, extract, 40min, lets cool, and mends weight, filtering, filtrate, with 30ml petroleum ether extraction 2 times, discards petroleum ether, shake by ethyl acetate and carry 2 times, each 15ml, merging filtrate, water bath method, residue adds 10ml60% methanol solution and dissolves, and filters, supply weight, 0.45um filtering with microporous membrane, to obtain final product.
Preferably, the method for building up of described JIESHITONG PIAN finger printing, comprise the following steps:
(1) reference substance solution is prepared: take Schaftoside reference substance, accurately weighed, add methanol and make every ml solution containing Schaftoside 0.05mg, to obtain final product；
(2) prepare need testing solution: JIESHITONG PIAN powder 1g, add 60% methanol 30ml, reflux, extract, 40min, let cool, mend weight, filter, filtrate, with 30ml petroleum ether extraction 2 times, discards petroleum ether, shakes by ethyl acetate and carry 2 times, each 15ml, merging filtrate, water bath method, residue adds 10ml60% methanol solution and dissolves, and filters, supplies weight, 0.45um filtering with microporous membrane, to obtain final product；
(3) measure: precision absorption reference substance solution injects high performance liquid chromatograph with each 10ul of need testing solution respectively, is measured according to following condition, records chromatogram, obtains JIESHITONG PIAN finger printing；
Wherein, chromatographic condition includes:
Chromatographic column: with octadecylsilane chemically bonded silica for filler；
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.2% phosphate aqueous solution, and mobile phase C is methanol；
Adopting gradient elution, elution program is as follows, and wherein mobile phase ratio is volume basis specific volume:
0～10min, mobile phase A is 10%, and Mobile phase B is 85%；Mobile phase C is 5%；
10～20min, mobile phase A is 10%；Mobile phase B is 85% → 82%；Mobile phase C is 5% → 8%；
20～30min, mobile phase A is 10% → 12%；Mobile phase B is 82% → 80%；Mobile phase C is 8%；
30～60min, mobile phase A is 12% → 17%；Mobile phase B is 80% → 75%；Mobile phase C is 8%；
Detection wavelength is 272；
Theoretical cam curve calculates by Schaftoside and is not less than 3000.
The method for building up of described JIESHITONG PIAN finger printing, also includes: multiple JIESHITONG PIAN finger printing are compared, selects common characteristic peak, obtain JIESHITONG PIAN characteristic fingerprint pattern.
nullThe method for building up of described JIESHITONG PIAN finger printing，It is in that in JIESHITONG PIAN finger printing or JIESHITONG PIAN characteristic fingerprint pattern and comprises 20 characteristic peaks，Each characteristic peak retention time is: peak 1:10.72min、Peak 2:11.35min、Peak 3:14.44min、Peak 4:15.36min、Peak 5:19.58min、Peak 6:20.56min、Peak 7:21.18min、Peak 8:22.43min、Peak 9:25.68min、Peak 10:26.57min、Peak 11:27.22min、Peak 12:29.35min、Peak 13:30.44min、Peak 14:31.36min、Peak 15:34.58min、Peak 16:35.56min、Peak 17:39.18min、Peak 18:43.43min、Peak 19:45.68min、Peak 20:50.57min.
nullThe method for building up of described JIESHITONG PIAN finger printing，It is in that in JIESHITONG PIAN finger printing or JIESHITONG PIAN characteristic fingerprint pattern，With Schaftoside for reference to peak，The relative retention time of each characteristic peak is: peak 1:0.5438、Peak 2:0.5642、Peak 3:0.7514、Peak 4:0.7907、Peak 5:1、Peak 6:1.0686、Peak 7:1.0891、Peak 8:1.1732、Peak 9:1.3583、Peak 10:1.3812、Peak 11:1.4547、Peak 12:1.5358、Peak 13:1.5742、Peak 14:1.6397、Peak 15:1.7273、Peak 16:1.7909、Peak 17:1.9850、Peak 18:2.1555、Peak 19:2.2651、Peak 20:2.5118
In described JIESHITONG PIAN finger printing or JIESHITONG PIAN characteristic fingerprint pattern, No. 5 cutting edges of a knife or a sword are Schaftoside comparison peak.
The contrast of chromatographic condition
(1) chromatographic column: compare the conventional chromatographic columns such as C18 post, phenyl post, nh 2 column, it has been found that composition in JIESHITONG PIAN is retained extreme difference with phenyl post by C8 post, it is impossible to reflection JIESHITONG PIAN composition comprehensively.Nh 2 column is forward distribution system, is not suitable for this experiment.Select C18 post to be easily separated, obtain good effect.
(2) mobile phase: find by studying, use merely acetonitrile-phosphoric acid water system or methanol-phosphoric acid water system, its sample solution does not go out peak or to go out peak little, the inferior separating effect of above-mentioned system, composition in medicine cannot be separated as much as possible, thus being difficult to differentiate according to its collection of illustrative plates, simultaneously because compositional polarity difference in calculus Tongli face is bigger, use unitary system ratio can not look after whole compositions, simple use water does inorganic phase, inferior separating effect, and peak shape is ugly, retention time fluctuation is relatively big, and what can not meet characteristic spectrum sets up requirement at all.In conjunction with the feature at the peak of non-principal component in the mobile phase of prior art and the JIESHITONG PIAN of separation thereof, by creative work, finally determine the ternary system of use acetonitrile-phosphoric acid water-methanol, under this condition, each chromatographic peak peak shape is sharp-pointed, and separating degree is better, and retention time is moderate.
(3) detection wavelength: by DAD chromatographic scan technology, the present invention have chosen 272nm as detection wavelength, and at this wavelength, chromatography is more, and baseline is steady, and each peak separating degree is better.
The present invention also provides for the authentication method of a kind of JIESHITONG PIAN, the method include by according to said method set up testing sample finger printing or characteristic fingerprint pattern and according to said method set up standard finger-print or characteristic fingerprint figure compare, to discern the false from the genuine.
JIESHITONG PIAN quality control: utilize Chinese medicine fingerprint area of computer aided similarity evaluation software, JIESHITONG PIAN finger printing and standard finger-print are compared, JIESHITONG PIAN finger printing calculates similarity with 20 characteristic peaks and is not less than the JIESHITONG PIAN of 0.8 for up-to-standard, JIESHITONG PIAN is made as a whole treating by the present invention, the nuance between different batches can be found out by comparing its characteristic peak, be suitable to the discriminating to JIESHITONG PIAN quality and control.
The invention have the advantage that
1, in existing " the Sanitation Ministry medicine standard Traditional Chinese medicine historical preparation ", JIESHITONG PIAN quality standard only has character, inspection project, it is impossible to effective control JIESHITONG PIAN quality.Fingerprint pattern technology is one of key technology of the modernization of Chinese medicine, it it is the state-of-the-art technology of Chinese medicine quality control, we use fingerprint pattern technology effectively to control the quality of JIESHITONG PIAN, compensate for the deficiency of existing Quality Control Technology, as long as selective preparation occurs the finger printing specified by the application, JIESHITONG PIAN can be confirmed as, so that it is guaranteed that the effectiveness of JIESHITONG PIAN, safety.
2, high performance liquid chromatography is adopted to determine in the process of JIESHITONG PIAN finger printing, selection by mobile phase, separation condition and parameter, final choice acetonitrile organic facies, the gradient peak owing to graded causes can be reduced, the phosphoric acid selecting Volume fraction to be 0.2 is that Mobile phase B is adjusted forward position, peak, mobile phase ratio is changed by concrete temporal sequence, the design parameter of above procedure cooperates, make target peak all can have with impurity peaks well to separate, simultaneously can well corresponding and metastable retention time, thus obtaining a good characteristic spectrum.
3, the present invention adopts JIESHITONG PIAN finger printing as the quality control method of JIESHITONG PIAN preparation, both the one-sidedness judging preparation total quality because only measuring one, two chemical compositions had been avoided, decrease again the probability artificially processed for requisite quality, the present invention is that quality complete, accurate evaluation JIESHITONG PIAN provides effective ways, so that the quality of product and curative effect are guaranteed.
4, the present invention has simplicity, it is easy to grasp, stable, and precision is high, the feature of favorable reproducibility.
Accompanying drawing explanation
Fig. 1 is the finger printing of 20 characteristic peaks of JIESHITONG PIAN described in embodiment 1.
Fig. 2 is JIESHITONG PIAN ten batch sample finger printing in embodiment 2.
Detailed description of the invention
Embodiment 1: JIESHITONG PIAN HPLC characteristic spectrum sets up experiment
1. instrument, reagent and test sample
Instrument: high performance liquid chromatograph: waterse2695 pump, UV2998 detector
Electronic balance: CP214 Ao Haosi instrument
Reference substance: Schaftoside (Wei Keqi bio tech ltd of Sichuan Province system, HPLC is more than 98), lot number: 121015
Sample: JIESHITONG PIAN (offer of Guangxi Wuzhou Pharmaceutical (group) limited company), lot number is respectively as follows: 140603,140204,140605,140703,140704,140705,140706,140707,140802,140803,140805
2. chromatographic condition
With octadecylsilane chemically bonded silica for filler；With acetonitrile for mobile phase A；With 0.2% phosphate aqueous solution for Mobile phase B；With methanol for mobile phase C, temperature is room temperature, and flow velocity is 1.0ml/min, carries out gradient elution by following procedure；Detection wavelength is 272nm；Theoretical cam curve should be not less than 3000 by Schaftoside；
0～10min, mobile phase A is 10%, and Mobile phase B is 85%；Mobile phase C is 5%；
10～20min, mobile phase A is 10%；Mobile phase B is 85% → 82%；Mobile phase C is 5% → 8%；
20～30min, mobile phase A is 10% → 12%；Mobile phase B is 82% → 80%；Mobile phase C is 8%；
30～60min, mobile phase A is 12% → 17%；Mobile phase B is 80% → 75%；Mobile phase C is 8%.
That is, in 0 minute to 10 minutes, mobile phase A volume ratio is 10%, and Mobile phase B volume ratio is 85%；Mobile phase C volume ratio is 5%；In 10 minutes to 20 minutes, mobile phase A volume ratio is 10%；Mobile phase B volume ratio is by 85% gradual change to volume ratio 82%；Mobile phase C volume ratio is by 5% gradual change to volume ratio 8%；In 20 minutes to 30 minutes, mobile phase A volume ratio is by 10% gradual change to volume ratio 12%；Mobile phase B volume ratio is by 82% gradual change to volume ratio 80%；Mobile phase C volume ratio is 8%；In 30 minutes to 60 minutes, mobile phase A volume ratio is by 12% gradual change to volume ratio 17%；Mobile phase B volume ratio is by 80% gradual change to volume ratio 75%；Mobile phase C volume ratio is 8%.
3. the preparation of reference substance solution
Take Schaftoside reference substance, accurately weighed, add methanol and make every ml solution containing Schaftoside 0.05mg, to obtain final product.
4. the preparation of need testing solution: JIESHITONG PIAN powder 1g, adds 60% methanol 30ml, reflux, extract, 40min, let cool, mend weight, filter, filtrate, with 30ml petroleum ether extraction 2 times, discards petroleum ether, shakes by ethyl acetate and carry 2 times, each 15ml, merging filtrate, water bath method, residue adds 10ml60% methanol solution and dissolves, and filters, supplies weight, 0.45um filtering with microporous membrane, to obtain final product.
5. the formulation of JIESHITONG PIAN standard finger-print
Taking JIESHITONG PIAN 10 batches, prepare test sample according to the preparation method of need testing solution, under above-mentioned liquid phase chromatogram condition, precision draws object of reference solution and each 10ul of need testing solution respectively, injects chromatograph of liquid, records chromatogram.According to the finger printing of 10 batch samples, formulate characteristic spectrum.Should presenting 20 characteristic peaks in test sample characteristic pattern, wherein 1 peak should be consistent with corresponding reference substance peak retention time；With Schaftoside reference peak for S peak, calculate the relative retention time of each characteristic peak and S peak, it is shown that the RSD of the relative retention time at each common characteristic peak is respectively less than 2%, meet fingerprint map analyzing requirement.According to standard finger-print, relative retention time setting is peak 1:0.5438, peak 2:0.5642, peak 3:0.7514, peak 4:0.7907, peak 5:1, peak 6:1.0686, peak 7:1.0891, peak 8:1.1732, peak 9:1.3583, peak 10:1.3812, peak 11:1.4547, peak 12:1.5358, peak 13:1.5742, peak 14:1.6397, peak 15:1.7273, peak 16:1.7909, peak 17:1.9850, peak 18:2.1555, peak 19:2.2651, peak 20:2.5118.In 20 characteristic peaks, the peak that No. 5 peaks are corresponding with Schaftoside, its retention time is consistent.
Table 1 common characteristic peak relative retention time table
| Peak number | Relative retention time |
| 1 | 0.5438 |
| 2 | 0.5642 |
| 3 | 0.7514 |
| 4 | 0.7907 |
| 5 | 1 |
| 6 | 1.0686 |
| 7 | 1.0891 |
| 8 | 1.1732 |
| 9 | 1.3583 |
| 10 | 1.3812 |
| 11 | 1.4547 |
| 12 | 1.5328 |
| 13 | 1.5742 |
| 14 | 1.6397 |
| 15 | 1.7273 |
| 16 | 1.7909 |
| 17 | 1.985 |
| 18 | 2.1555 |
| 19 | 2.2651 |
| 20 | 2.5118 |
Embodiment 2: JIESHITONG PIAN HPLC characteristic spectrum is set up
JIESHITONG PIAN is provided by Guangxi Wuzhou Pharmaceutical (group) limited company.
Measure according to high performance liquid chromatography (one annex VID of " Chinese Pharmacopoeia " version in 2010):
With octadecylsilane chemically bonded silica for filler；With acetonitrile for mobile phase A；With 0.2% phosphate aqueous solution for Mobile phase B；With methanol for mobile phase C, temperature is room temperature, and flow velocity is 1.0ml/min, carries out gradient elution by following procedure；Detection wavelength is 272nm；Theoretical cam curve should be not less than 3000 by Schaftoside；
0～10min, mobile phase A is 10%, and Mobile phase B is 85%；Mobile phase C is 5%；
10～20min, mobile phase A is 10%；Mobile phase B is 85% → 82%；Mobile phase C is 5% → 8%；
20～30min, mobile phase A is 10% → 12%；Mobile phase B is 82% → 80%；Mobile phase C is 8%；
30～60min, mobile phase A is 12% → 17%；Mobile phase B is 80% → 75%；Mobile phase C is 8%；
That is, in 0 minute to 10 minutes, mobile phase A volume ratio is 10%, and Mobile phase B volume ratio is 85%；Mobile phase C volume ratio is 5%；In 10 minutes to 20 minutes, mobile phase A volume ratio is 10%；Mobile phase B volume ratio is by 85% gradual change to volume ratio 82%；Mobile phase C volume ratio is by 5% gradual change to volume ratio 8%；In 20 minutes to 30 minutes, mobile phase A volume ratio is by 10% gradual change to volume ratio 12%；Mobile phase B volume ratio is by 82% gradual change to volume ratio 80%；Mobile phase C volume ratio is 8%；In 30 minutes to 60 minutes, mobile phase A volume ratio is by 12% gradual change to volume ratio 17%；Mobile phase B volume ratio is by 80% gradual change to volume ratio 75%；Mobile phase C volume ratio is 8%.
The preparation of reference substance solution: take Schaftoside reference substance, accurately weighed, add methanol and make every ml solution containing Schaftoside 0.05mg, to obtain final product.
The preparation of need testing solution: JIESHITONG PIAN powder 1g, adds 60% methanol 30ml, reflux, extract, 40min, let cool, mend weight, filter, filtrate, with 30ml petroleum ether extraction 2 times, discards petroleum ether, shakes by ethyl acetate and carry 2 times, each 15ml, merging filtrate, water bath method, residue adds 10ml60% methanol solution and dissolves, and filters, supplies weight, 0.45um filtering with microporous membrane, to obtain final product.
Algoscopy: precision draws object of reference solution and each 10ul of need testing solution respectively, injects chromatograph of liquid, records chromatogram.
Should presenting 20 characteristic peaks in test sample characteristic pattern, wherein 1 peak should be consistent with corresponding reference substance peak retention time；With Schaftoside reference peak for S peak, calculate the relative retention time of each characteristic peak and S peak, it is shown that the RSD of the relative retention time at each common characteristic peak is respectively less than 2%, meet fingerprint map analyzing requirement.According to standard finger-print, relative retention time setting is peak 1:0.5438, peak 2:0.5642, peak 3:0.7514, peak 4:0.7907, peak 5:1, peak 6:1.0686, peak 7:1.0891, peak 8:1.1732, peak 9:1.3583, peak 10:1.3812, peak 11:1.4547, peak 12:1.5358, peak 13:1.5742, peak 14:1.6397, peak 15:1.7273, peak 16:1.7909, peak 17:1.9850, peak 18:2.1555, peak 19:2.2651, peak 20:2.5118.In 20 characteristic peaks, the peak that No. 5 peaks are corresponding with Schaftoside, its retention time is consistent.
Claims (9)
1. the method for building up of a JIESHITONG PIAN finger printing, it is characterised in that: the method includes adopting high performance liquid chromatography that JIESHITONG PIAN is detected, and wherein chromatographic condition includes；
Chromatographic column: with octadecylsilane chemically bonded silica for filler；
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.2% phosphate aqueous solution, and mobile phase C is methanol；
Adopting gradient elution, elution program is as follows, and wherein mobile phase ratio is volume basis specific volume:
0～10min, mobile phase A is 10%, and Mobile phase B is 85%；Mobile phase C is 5%；
10～20min, mobile phase A is 10%；Mobile phase B is 85% → 82%；Mobile phase C is 5% → 8%；
20～30min, mobile phase A is 10% → 12%；Mobile phase B is 82% → 80%；Mobile phase C is 8%；
30～60min, mobile phase A is 12% → 17%；Mobile phase B is 80% → 75%；Mobile phase C is 8%；
Detection wavelength is 272；
Theoretical cam curve calculates by Schaftoside and is not less than 3000.
2. method according to claim 1, it is characterised in that described method also includes preparing reference substance solution by following steps: take Schaftoside reference substance, accurately weighed, adds methanol and makes every ml solution containing Schaftoside 0.05mg, to obtain final product.
3. method according to claim 1, it is characterised in that described method also includes preparing need testing solution by following steps: JIESHITONG PIAN powder 1g, adds 60% methanol 30ml, reflux, extract, 40min, lets cool, and mends weight, filtering, filtrate, with 30ml petroleum ether extraction 2 times, discards petroleum ether, shake by ethyl acetate and carry 2 times, each 15ml, merging filtrate, water bath method, residue adds 10ml60% methanol solution and dissolves, and filters, supply weight, 0.45um filtering with microporous membrane, to obtain final product.
4. the method according to claim 1-3, it is characterised in that said method comprising the steps of:
(1) reference substance solution is prepared: take Schaftoside reference substance, accurately weighed, add methanol and make every ml solution containing Schaftoside 0.05mg, to obtain final product；
(2) prepare need testing solution: JIESHITONG PIAN powder 1g, add 60% methanol 30ml, reflux, extract, 40min, let cool, mend weight, filter, filtrate, with 30ml petroleum ether extraction 2 times, discards petroleum ether, shakes by ethyl acetate and carry 2 times, each 15ml, merging filtrate, water bath method, residue adds 10ml60% methanol solution and dissolves, and filters, supplies weight, 0.45um filtering with microporous membrane, to obtain final product；
(3) measure: precision absorption reference substance solution injects high performance liquid chromatograph with each 10ul of need testing solution respectively, is measured according to following condition, records chromatogram, obtains JIESHITONG PIAN finger printing；
Wherein, chromatographic condition includes:
Chromatographic column: with octadecylsilane chemically bonded silica for filler；
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 0.2% phosphate aqueous solution, and mobile phase C is methanol；
Adopting gradient elution, elution program is as follows, and wherein mobile phase ratio is volume basis specific volume:
0～10min, mobile phase A is 10%, and Mobile phase B is 85%；Mobile phase C is 5%；
10～20min, mobile phase A is 10%；Mobile phase B is 85% → 82%；Mobile phase C is 5% → 8%；
20～30min, mobile phase A is 10% → 12%；Mobile phase B is 82% → 80%；Mobile phase C is 8%；
30～60min, mobile phase A is 12% → 17%；Mobile phase B is 80% → 75%；Mobile phase C is 8%；
Detection wavelength is 272；
Theoretical cam curve calculates by Schaftoside and is not less than 3000.
5. the method according to any one of claim 1-4, it is characterised in that described method also includes: multiple JIESHITONG PIAN finger printing are compared, selects common characteristic peak, obtain JIESHITONG PIAN characteristic fingerprint pattern.
null6. method according to claim 5，It is characterized in that，Described JIESHITONG PIAN finger printing or JIESHITONG PIAN characteristic fingerprint pattern comprise 20 characteristic peaks，Each characteristic peak retention time is: peak 1:10.72min、Peak 2:11.35min、Peak 3:14.44min、Peak 4:15.36min、Peak 5:19.58min、Peak 6:20.56min、Peak 7:21.18min、Peak 8:22.43min、Peak 9:25.68min、Peak 10:26.57min、Peak 11:27.22min、Peak 12:29.35min、Peak 13:30.44min、Peak 14:31.36min、Peak 15:34.58min、Peak 16:35.56min、Peak 17:39.18min、Peak 18:43.43min、Peak 19:45.68min、Peak 20:50.57min.
null7. method according to claim 6，It is characterized in that，In described JIESHITONG PIAN finger printing or JIESHITONG PIAN characteristic fingerprint pattern，With Schaftoside for reference to peak，The relative retention time calculating each characteristic peak is: peak 1:0.5438、Peak 2:0.5642、Peak 3:0.7514、Peak 4:0.7907、Peak 5:1、Peak 6:1.0686、Peak 7:1.0891、Peak 8:1.1732、Peak 9:1.3583、Peak 10:1.3812、Peak 11:1.4547、Peak 12:1.5358、Peak 13:1.5742、Peak 14:1.6397、Peak 15:1.7273、Peak 16:1.7909、Peak 17:1.9850、Peak 18:2.1555、Peak 19:2.2651、Peak 20:2.5118.
8. method according to claim 7, it is characterised in that in described JIESHITONG PIAN finger printing or JIESHITONG PIAN characteristic fingerprint pattern, peak 5 compares peak for Schaftoside.
9. an authentication method for JIESHITONG PIAN, the method will according to any one of claim 1 to 8
Method sets up testing sample finger printing or characteristic fingerprint pattern and standard finger-print or characteristic fingerprint figure according to said method foundation compare.
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| CN201511022764.1A CN105738547B (en) | 2015-12-31 | 2015-12-31 | A kind of method for building up of JIESHITONG PIAN finger-print |
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| CN110441443A (en) * | 2019-09-20 | 2019-11-12 | 广东一方制药有限公司 | A kind of pyrrosia petiolosaChing, pyrrosia lingua, pyrrosia sheareriChing and North China pyrrosia lingua UPLC characteristic spectrum construction method and its discrimination method |
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| CN110441443B (en) * | 2019-09-20 | 2022-06-24 | 广东一方制药有限公司 | UPLC characteristic spectrum construction method and identification method of pyrrosia peduncularis, pyrrosia lingua, pyrrosia cottonii and pyrrosia huabeiensis |
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