CN105938125B - The HPLC fingerprint atlas detection methods of Radix Polygoni Multiflori - Google Patents
The HPLC fingerprint atlas detection methods of Radix Polygoni Multiflori Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses the HPLC fingerprint atlas detection methods of Radix Polygoni Multiflori, it includes the following steps：(1) preparation of reference substance solution；(2) preparation of reference solution：(3) foundation of reference fingerprint：(4) preparation of test solution；(5) detection of test solution.The finger-print appearance of the present invention is more, and separating degree is good, reproducible, and precision is high, and effective guarantee is provided for overall monitor Radix Polygoni Multiflori quality.Using the extracting method and chromatographic condition of fingerprint atlas detection method of the present invention, the content of 16 kinds of ingredients in Radix Polygoni Multiflori can also be measured, so as to control the quality of Radix Polygoni Multiflori from more angles.
Description
Technical field
The present invention relates to the detections of the HPLC finger-prints of Control of drug quality method and technology field more particularly to Radix Polygoni Multiflori
Method.
Background technology
The fleece-flower root is the dried root of polygonum multiflorum thunb (Polygonum Multiflorum Thunb.), property
Flat, sweet in flavor, bitter, the thoughts of returning home, liver, large intestine channel have effects that removing toxic substances, the carbuncle that disappears, preventing malaria, relax bowel；For conventional Chinese medicine.It is complete
State various regions are widely distributed, and main product is in Henan, Hubei, Guangxi, Guangdong, Guizhou, Sichuan and other places.
Talan glycoside and anthraquinone component in the fleece-flower root are its representative active constituent.Wherein, Stibene-glucoside
With significant neuroprotection；Anthraquinone component has the works such as anti-inflammatory, resisting pathogenic microbes, antitumor, diuresis, liver protection
With wherein rheum emodin has the effects that anti-inflammatory, resisting pathogenic microbes, antitumor, diuresis, liver protection, rheum emodin -8- β-o- pyrans Portugal
Polyglycoside then has nootropic activity.
At present《Chinese Pharmacopoeia》Using UV-visible spectrophotometry to the 2 of the fleece-flower root under version fleece-flower root items in 2015,
3,5,4- tetrahydroxystilbene -2-O- β-D glucosides and emodin content are determined, and matter is established with measurement result
Amount standard.
However, it is contemplated that the ingredient of Chinese medicine is various, and is easy to influence each other between each ingredient, only with one or two effectively at
The content divided is difficult to the real quality of thoroughly evaluating Chinese medicine.Therefore, need at present one kind can monitor in Radix Polygoni Multiflori it is multiple at
The detection method divided, to monitor the quality condition of Radix Polygoni Multiflori more fully hereinafter.
Invention content
To solve the above problems, the present invention provides a kind of HPLC fingerprint atlas detection methods of Radix Polygoni Multiflori, it includes
Following steps：
The HPLC fingerprint atlas detection methods of Radix Polygoni Multiflori, it is characterised in that：It includes the following steps：
(1) preparation of reference substance solution：Radix Polygoni Multiflori medicinal material is taken, medicinal powder, 40~60% ethanol waters are crushed to obtain
Extraction, filtering, obtains Radix Polygoni Multiflori reference substance solution；Preferred concentration of alcohol is 50%；
(2) preparation of reference solution：Take gallic acid, procyanidine B1, catechin, gallate, aloe rheum officinale
It is plain glycosides, polygonin, Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, big
Yellow acid, Chrysophanol, rheum emodin, Physcion reference substance are appropriate, and mixing is added methanol, obtains reference solution；
(3) foundation of reference fingerprint：
The reference substance solution for taking reference solution respectively and being prepared by different batches Radix Polygoni Multiflori medicinal material, injection are efficient
Liquid chromatograph detects, and obtains the chromatographic data of each batch medicinal material and object of reference；Chromatographic condition is as follows：
Detection wavelength：254~275nm；
Chromatographic column：C18 chromatographic columns and C18 pre-columns；
Mobile phase：Mobile phase A is 0.1% formic acid water, and Mobile phase B is acetonitrile；
Gradient elution program is as follows：
Gained chromatographic data is handled, the reference fingerprint of Radix Polygoni Multiflori is established；
(4) preparation of test solution：Untested medicinal material is taken, it is molten that test sample is prepared according to the identical method of step (1)
Liquid；
(5) according to the identical chromatographic condition of step (3), by test solution injection high performance liquid chromatograph detection, by institute
It obtains chromatogram to be compared with reference fingerprint, Radix Polygoni Multiflori and its quality condition can be identified.
Further, in step (1), the extraction ultrasonic extraction.
Further, the time of the ultrasonic extraction is 60min.
Further, in step (1), the temperature of the extraction is 40 DEG C.
Further, in step (1), the w/v of the medicinal material ethanol water is 1g:20mL.
Further, the C18 chromatographic columns are ZORBAX C18 chromatographic columns, length 250mm, internal diameter 4.6mm, filler
Grain size be 5 μm；The C18 pre-columns are ZORBAX C18 chromatographic columns, length 12.5mm, internal diameter 4.6mm, the grain size of filler
For 5 μm of.
Further, the column temperature of the chromatographic condition is 30 DEG C.
Further, the flow velocity of the chromatographic condition is 1.0mL/min.
Further, the reference fingerprint is as shown in Figure 5.
The present invention also provides method that is a kind of while measuring 16 kinds of ingredients in Radix Polygoni Multiflori, 16 kinds of ingredients are not eat
Sub- acid, procyanidine B1, catechin, gallate, Barbaloin, polygonin, Stibene-glucoside, rheochrysin, white lamb's-quarters
Reed alcohol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, Rhein, Chrysophanol, rheum emodin and Physcion；
It includes the following steps：
A, the foundation of 16 kinds of ingredient standard curves：
16 kinds of ingredient mixed reference substance solutions are taken, are detected according to the identical chromatographic condition of step (3) in aforementioned detection method,
Obtain 16 kinds of respective standard curves of ingredient；
B, Radix Polygoni Multiflori medicinal material is taken, according to the identical method of step (1) in aforementioned detection method, it is molten that test sample is prepared
Liquid injects high performance liquid chromatography, is detected with the identical chromatographic conditions of step a, Radix Polygoni Multiflori is obtained according to the standard curve of step a
In 16 kinds of ingredients content.
The finger-print appearance of the present invention is more, separating degree is good, reproducible, precision is high, and for overall monitor life, what is first
The material amount of crow provides effective guarantee.
Meanwhile using the extracting method and chromatographic condition of fingerprint atlas detection method of the present invention, life can also be measured simultaneously
16 kinds of ingredient-gallic acids, procyanidine B in the fleece-flower root1, catechin, gallate, Barbaloin, polygonin,
Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, Rhein, Chrysophanol,
The content of rheum emodin, Physcion, so as to from the quality of medicinal material of more angle monitoring Radix Polygoni Multifloris.
Obviously, the above according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific implementation mode of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Description of the drawings
Fig. 1 is the finger-print (A-F) of Radix Polygoni Multiflori difference gradient elution program.
Fig. 2 is the finger-print (A-D) of ultrasound 60min under Radix Polygoni Multiflori condition of different temperatures.
Fig. 3 is the finger-print (A-E) under the Radix Polygoni Multiflori difference ultrasonic extraction time.
Fig. 4 is the standard items collection of illustrative plates of 16 ingredients of the fleece-flower root：1：Gallic acid, 2：Proanthocyanidin B1,3：Catechin, 4：Not yet
Infanticide acid esters, 5：Aloe rheochrysin, 6：Polygonin, 7：Stibene-glucoside, 8：Ponticin, 9：Resveratrol, 10：Rheum emodin
Glycosides, 11：Physcion glycosides, 12：Aloe-emodin, 13：Rhein, 14：Rheum emodin, 15：Chrysophanol, 16：Physcion.
Fig. 5 is the reference fingerprint of the fleece-flower root
Fig. 6 is the finger-print of 25 batches of commercially available Radix Polygoni Multifloris.
Specific implementation mode
Reagent
Gallic acid (lot number：150226)；Procyanidine B1(lot number：140628)；Catechin (lot number：141224)；Not yet
Infanticide acid esters (lot number：140730)；Aloe-emodin -8-O- β-D-Glucose glycosides are (referred to as：Barbaloin, lot number：
141225)；Polygonin (lot number：141228)；2,3,5,4- tetrahydroxystilbenes -2-O- β-D-Glucose glycosides are (referred to as：Hexichol
Ethylene glycosides, lot number：141121)；Ponticin (lot number：141209)；Resveratrol (lot number：150122)；Rheum emodin -8- β-O-
Glucopyranoside is (referred to as：Rheum emodin glycosides, lot number：141209)；Physcion -8-O- β-D-Glucose glycosides are (referred to as：Rheum officinale
Plain methyl ether glycosides, lot number：150120)；Chrysophanol (lot number：140325) it purchases in Chengdu Crow agate biology Co., Ltd.Aloe
Rheum emodin (lot number：MUST-10112301)；Rhein (lot number：MUST-11032801)；Rheum emodin (lot number：MUST-
12022715)；Physcion (lot number：MUST-12022005 it) purchases in the bio tech ltd Man Site.
Medicinal material
25 batches of Radix Polygoni Multiflori medicinal materials are accredited as polygonaceae plant Polygonum through Chengdu University of Traditional Chinese Medicine's Pharmacognosy Studies room
The dried root of multiflorum Thunb..Polygonum multiflorum medicinal material sample source is shown in Table 1.
1 polygonum multiflorum medicinal material sample of table
Laboratory apparatus
1260 high performance liquid chromatograph devices of Agilent (pump G1312B, DEACB07224；Injector G1329B,
DEAAC25245；Column oven G1316A, DEACN27098；UV detector G4212BDEAA307363)；KQ-500DB ultrasonic waves
Washer (Kunshan Ultrasonic Instruments Co., Ltd.)；11D types electronic analytical balance (Sartorius companies)；BP-2Vortex-
2 turbula shakers of Genie (Scientific Industries, the U.S.)；TG16-WS types table model high speed centrifuge (Hunan instrument from
Scheming Co., Ltd)；Vacuum diaphragm pump (Tianjin Jin Teng experimental facilities Co., Ltd).
Experiment reagent
Absolute ethyl alcohol (lot number：20120306) it purchases in Chengdu chemical industry near a river.Formic acid, acetonitrile, water are chromatographic grade, are purchased from
Fisher companies, other reagents are that analysis is pure.
The screening of 1 experiment condition of embodiment
1. mobile phase conditional filtering
Under the same sample sampling condition of Radix Polygoni Multiflori, 0.1% formic acid water and acetonitrile are pumped as the A of elution mobile phase respectively
It is pumped with B, condition of gradient elution screening is carried out with A and B different volumes ratios (v/v) in different time periods, gradient elution program is shown in
Table 2 obtains corresponding Radix Polygoni Multiflori finger-print and sees Fig. 1 (A-F), and as shown in Figure 1, Figure 1A fails to separate substantially；Figure 1B, C, D
Middle part-time point collection of illustrative plates fails to separate；Time segment baseline drift is serious in the middle part of Fig. 1 E, and it is good that each ingredient is showed in Fig. 1 F
Separation, baseline is substantially steady, therefore the condition of gradient elution is set as to the fingerprint chromatogram analysis condition of Radix Polygoni Multiflori.
The 2 gradient elution time of table (T) changes and the variation of water phase (A pumps) ratio
2. temperature is screened when Radix Polygoni Multiflori extracts
4 parts of the Radix Polygoni Multiflori sample of phase homogenous quantities is taken, 50% ethanol water of 20 times of quality is added, after impregnating 30min, respectively
Ultrasound 60min, sample introduction after filtering are established under the gradient elution program screened under the conditions of 20 DEG C, 40 DEG C, 60 DEG C, 80 DEG C
The finger-print of the Radix Polygoni Multiflori of different Extracting temperatures is shown in Fig. 2 (A-D).Record Stibene-glucoside at each temperature, rheum emodin glycosides,
Physcion glycosides obtains table 3 with rheum emodin peak area using the variation of this 4 kinds of ingredient covers product as screening conditions.In conjunction with Fig. 2
Under the conditions of understanding 40 DEG C with table 3, the peak area of 4 kinds of ingredients is maximum, prompts the component content highest obtained under this condition, therefore
It is 40 DEG C that Extracting temperature, which is arranged,.
Ultrasound 60min obtains the peak area of 4 kinds of ingredients under 3 condition of different temperatures of table
3. Radix Polygoni Multiflori extraction time screens
5 parts of the Radix Polygoni Multiflori sample of phase homogenous quantities is taken, 50% ethanol water of 20 times of quality is added, after impregnating 30min, is being sieved
Ultrasound 10min, 20min, 40min, 60min and 80min under the conditions of selecting obtain 40 DEG C, sample introduction after filtering, what is screened
The finger-print that the Radix Polygoni Multiflori of different Extracting temperatures is established under gradient elution program is shown in Fig. 4 (A-E).Record two at each temperature
With rheum emodin peak area, the variation with this 4 kinds of ingredient covers product is screening item for styrene glycosides, rheum emodin glycosides, Physcion glycosides
Part obtains table 4.According to Fig. 4 and table 4 as time went on, the peak area of each ingredient increases, and peak area when 60min reaches
To maximum, increase no longer at any time and increase, the component content highest for prompting ultrasound 60min can be obtained, therefore be arranged and carry
It is 40 DEG C to take temperature, ultrasonic time 60min.
Ultrasonic different time obtains the peak area of 4 kinds of ingredients under the conditions of 4 40 DEG C of table
The method and its methodology validation of 2 present invention of embodiment
The preparation of 1 sample solution
It is spare that 25 batches of commercially available Radix Polygoni Multifloris are broken into coarse powder respectively.Precision weighs each coarse powder 0.25g, sets in conical flask with stopper,
Add 20 times of 50% ethanol waters of amount, after impregnating 30min, ultrasonic extraction 60min at 40 DEG C, constant volume to 5mL.0.45 μm of micropore filter
Membrane filtration mistake takes subsequent filtrate sample introduction.
The preparation of 2.2 reference substance solutions
Precision weighs reference substance gallic acid, procyanidine B1, catechin, gallate, Barbaloin, giant knotweed
Glycosides, Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, Rhein, rheum officinale
Phenol, rheum emodin, Physcion reference substance are in 10mL volumetric flasks, methanol constant volume to scale, be configured to concentration be respectively 131,
98, the storing solution of 185,142,78,125,5400,142,94,750,86,71,81,500,25,88 μ g/mL, 4 DEG C of storages are standby
With.
2.3 chromatographic condition
Chromatographic column ZORBAX C18 (4.6mm*250mm, 5 μm), pre-column ZORBAX C18 (4.6mm*12.5mm, 5 μm)；Stream
Fast 1.0mL/min, 30 DEG C of column temperature, sample size 5 μ L, gradient elution A are 0.1% formic acid water, and B is acetonitrile, flow phase system proportioning
It is shown in Table 5.
5 condition of gradient elution of table
The foundation of 2.4 finger-prints
The traditional Chinese medicine fingerprint area of computer aided similarity evaluation software recommended using Chinese Pharmacopoeia Commission A editions carries out each
The similarity analysis of a concocted time sample.
3 experimental results
Fig. 4 is the liquid chromatogram of the standard items of 16 kinds of ingredients in Radix Polygoni Multiflori.
3.1 methodological study
3.1.1 precision
Prepare gallic acid, procyanidine B that concentration is followed successively by basic, normal, high 3 concentration1, catechin, gallate,
Barbaloin, polygonin, Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe are big
Flavine, Rhein, Chrysophanol, rheum emodin, Physcion quality-control sample, each concentration is 3 parts parallel, according to color under " 2.3 " item
Spectral condition is analyzed.Withinday precision, day to day precision for three days on end are investigated, peak area is measured and calculates each ingredient
RSD% values, are shown in Table 6.
Each ingredient of 6 fleece-flower root of table in a few days and day to day precision
As shown in Table 6, gallic acid, procyanidine B1, catechin, gallate, Barbaloin, polygonin,
Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, Rhein, Chrysophanol,
Each ingredient of rheum emodin, Physcion in a few days and day to day precision RSD% is respectively less than 6.00%, method precision is good.
3.1.2 reproducibility
It takes with a collection of 6 parts of Radix Polygoni Multiflori medicinal material, is prepared according to test solution preparation method under " 2.1 " item, under " 2.3 " item
Chromatographic condition is measured, and measures each ingredient gallic acid, procyanidine B in 6 parts of samples1, catechin, gallate, reed
Luxuriant growth rheum emodin glycosides, polygonin, Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe rheum officinale
The peak area of element, Rhein, Chrysophanol, rheum emodin, Physcion, standard curve calculate the concentration of each ingredient, calculate it
RSD% is shown in Table 7.
3.1.3 stability
It takes with a collection of Radix Polygoni Multiflori medicinal material, is prepared according to test solution preparation method under " 2.1 " item, with color under " 2.3 " item
Spectral condition is measured, respectively gallic acid, procyanidine B in 0,3,6,9,12 and sample introduction for 24 hours, determination sample1, catechu
It is element, gallate, Barbaloin, polygonin, Stibene-glucoside, ponticin, resveratrol, rheum emodin glucosides, big
The peak area of flavine methyl ether glycosides, aloe-emodin, Rhein, Chrysophanol, rheum emodin, Physcion, standard curve calculate each
The concentration of ingredient calculates RSD%, is shown in Table 7.
The reproducibility of each ingredient of 7 fleece-flower root of table, study on the stability
As shown in Table 7, each ingredient of the fleece-flower root, RSD% values are respectively less than 2.00%, meet 2015 editions States Pharmacopoeia specifications, show
The favorable reproducibility of each ingredient under the conditions of the extracting method, sample are good in 12h analysis times internal stability.
3.1.4 the rate of recovery
The same a collection of Radix Polygoni Multiflori medicinal material for taking known content, is divided into 2 parts, portion is added 50% isometric ethyl alcohol and is diluted to original
There is the 50% of concentration, gallic acid, the procyanidine B of known and close concentration is added in undiluted another1, catechin do not have
Infanticide acid esters, Barbaloin, polygonin, Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion
Glycosides, aloe-emodin, Rhein, Chrysophanol, rheum emodin, Physcion it is isometric, HPLC measure dilution after sample in
The peak area of each ingredient, substitutes into standard curve and is calculated, used after the concentration of each ingredient and isometric addition reference substance
C%=CSample+mark-CSample/CMark* 100%, calculation formula calculates the rate of recovery % of each ingredient, the rate of recovery of each ingredient of the fleece-flower root exists
Between 90%~110%, the rate of recovery is higher, the results are shown in Table 8.
The rate of recovery of each ingredient of 8 fleece-flower root of table is investigated
As shown in Table 8, each components recoveries value of the fleece-flower root shows between 90.00%~120.00% in the analysis
Method condition has the preferable rate of recovery.
3.1.5 prepared by each ingredient standard curve of the fleece-flower root
Gallic acid, procyanidine B1, catechin, gallate, aloe-emodin-glycosides, polygonin, talan
It is glycosides, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, Rhein, Chrysophanol, rheum emodin, big
Flavine methyl ether storing solution carries out gradient dilution, obtains a series of standard solution of concentration, by the liquid-phase condition under " 2.3 " item to each
The peak area of ingredient is measured, and the standard curve for obtaining each ingredient is shown in Table 9.
The standard curve and the range of linearity of each ingredient of 9 fleece-flower root of table
The assay of 3.2 25 batches of commercially available Radix Polygoni Multiflori ingredients
25 batches of commercially available Radix Polygoni Multifloris are measured by the preparation for carrying out sample under " 2.1 " item with chromatographic condition sample introduction under " 2.3 " item.
Each each sample replication 3 times, to be worth to gallic acid in sample, procyanidine B1, catechin, gallate,
Barbaloin, polygonin, Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe are big
The content of flavine, Rhein, Chrysophanol, rheum emodin, Physcion, the results are shown in Table 10.
3.3 fingerprint similarities calculate
The finger-print of 25 batches of what commercially available Radix Polygoni Multifloris is obtained through experiment, by the 25 batches of polygonum multiflorum medicinal material sample finger-prints
AIA formatted data files import traditional Chinese medicine fingerprint similarity evaluation system research version (2004A editions), carry out similarity calculation,
With the higher Daba Mountain (number of each component content：S5 it is with reference to collection of illustrative plates, using median method, time that) place of production, which is finger-print,
Window width is 0.1min, is matched using Supplements, obtains Radix Polygoni Multiflori reference fingerprint, sees that Fig. 5, area of computer aided are similar
Degree appraisal report the results are shown in Table 1.The finger-print of 25 batches of polygonum multiflorum medicinal material samples is shown in Fig. 6
The commercially available Radix Polygoni Multiflori similarity calculation result of 11 25 batches, table
The reference fingerprint and sample finger-print globality set up by the evaluation system of traditional Chinese medicine fingerprint
(retention time and peak area) carries out similarity system design, to which traditional Chinese medicine quality is evaluated and be controlled, to obtain consistency
And otherness.Similarity generally thinks to meet the requirements between 0.85~1.0.It can substantially find out each ingredient certain from Fig. 6
Retention time error range in (0.1min) can have with the reference fingerprint of generation it is preferable overlapping, similarity between
Between 0.95~0.99, and 1 and No. 13 medicinal material similarity is more than 0.85 as judgement between 0.45~0.70 using similarity
The qualified or not limit of polygonum multiflorum medicinal material, then 1 and No. 13 medicinal material be determined as unqualified Radix Polygoni Multiflori sample.
To sum up test result can be seen that the present invention finger-print appearance it is more, separating degree is good, reproducible, accurate
Degree is high, and effective guarantee is provided for the material amount of overall monitor Radix Polygoni Multiflori.
Meanwhile using the extracting method and chromatographic condition of fingerprint atlas detection method of the present invention, life can also be measured simultaneously
16 kinds of ingredients in the fleece-flower root --- gallic acid, procyanidine B1, catechin, gallate, Barbaloin, giant knotweed
Glycosides, Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, Rhein, rheum officinale
The content of phenol, rheum emodin, Physcion, so as to from the quality of medicinal material of more angle monitoring Radix Polygoni Multifloris.
Claims (11)
1. the HPLC fingerprint atlas detection methods of Radix Polygoni Multiflori, it is characterised in that：It includes the following steps：
(1) preparation of reference substance solution：Radix Polygoni Multiflori medicinal material is taken, medicinal powder is crushed to obtain, 40~60% ethanol waters extract,
Filtering, obtains Radix Polygoni Multiflori reference substance solution；
(2) preparation of reference solution：Take gallic acid, Proanthocyanidin B1, catechin, gallate, Barbaloin,
Polygonin, Stibene-glucoside, ponticin, resveratrol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, Rhein,
Chrysophanol, rheum emodin, Physcion reference substance are appropriate, and mixing is added methanol, obtains reference solution；
(3) foundation of reference fingerprint：
The reference substance solution for taking reference solution respectively and being prepared by different batches Radix Polygoni Multiflori medicinal material injects efficient liquid phase
Chromatograph detects, and obtains the chromatographic data of each batch medicinal material and object of reference；Chromatographic condition is as follows：
Detection wavelength：254~275nm;
Chromatographic column：C18 chromatographic columns and C18 pre-columns；
Mobile phase：Mobile phase A is 0 .1% formic acid waters, and Mobile phase B is acetonitrile；
Gradient elution program is as follows：
Gained chromatographic data is handled, the reference fingerprint of Radix Polygoni Multiflori is established；
(4) preparation of test solution：Untested medicinal material is taken, test solution is prepared according to the identical method of step (1)；
(5) according to the identical chromatographic condition of step (3), by test solution injection high performance liquid chromatograph detection, by gained color
Spectrogram is compared with control fingerprint image promise, can identify Radix Polygoni Multiflori.
2. detection method according to claim 1, it is characterised in that：In step (1), the extraction ultrasonic extraction；It is described
Concentration of alcohol is 50%.
3. detection method according to claim 2, it is characterised in that：The time of the ultrasonic extraction is 60min.
4. according to claim 1-3 any one of them detection methods, it is characterised in that：In step (1), the temperature of the extraction
It is 40 DEG C.
5. detection method according to claim 1, it is characterised in that：In step (1), the weight of the medicinal material ethanol water
Amount volume ratio is 1g：20mL.
6. detection method according to claim 1, it is characterised in that：The C18 chromatographic columns are ZORBAX C18 chromatographic columns,
Length is 250mm, and the grain size of internal diameter 4.6mm, filler are 5um；The C18 pre-columns are ZORBAX C18 chromatographic columns, and length is
The grain size of 12 .5mm, internal diameter 4.6mm, filler are 5um.
7. according to claim 1-3,5 any one of them detection methods, it is characterised in that：The column temperature of the chromatographic condition is 30
℃。
8. detection method according to claim 4, it is characterised in that：The column temperature of the chromatographic condition is 30 DEG C.
9. detection method according to claim 7, it is characterised in that：The flow velocity of the chromatographic condition is 1.0mL/min.
10. detection method according to claim 8, it is characterised in that：The flow velocity of the chromatographic condition is 1.0mL/min.
11. method that is a kind of while measuring 16 kinds of ingredients in Radix Polygoni Multiflori, it is characterised in that：16 kinds of ingredients are nutgall
Acid, Proanthocyanidin B1, catechin, gallate, Barbaloin, polygonin, Stibene-glucoside, rheochrysin, white black false hellebore
Alcohol, rheum emodin glycosides, Physcion glycosides, aloe-emodin, Rhein, Chrysophanol, rheum emodin and Physcion；
It includes the following steps：
A, the foundation of 16 kinds of ingredient standard curves：
16 kinds of ingredient mixed reference substance solutions are taken, according to the identical color of step (3) in any one of claim 1-10 detection methods
Spectral condition detects, and obtains 16 kinds of respective standard curves of ingredient；
B, Radix Polygoni Multiflori medicinal material is taken, according to the identical method of step (1) in any one of claim 1-10 detection methods, is prepared into
To test solution, high performance liquid chromatography is injected, is detected with the identical chromatographic conditions of step a, is obtained according to the standard curve of step a
To the content of 16 kinds of ingredients in Radix Polygoni Multiflori.
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| 36批何首乌药材质量的HPLC指纹图谱评价研究;武政等;《中国药学杂志》;20060228;第41卷(第4期);全文 * |
| 不同产地何首乌 HPLC 指纹图谱研究;李帅锋等;《中草药》;20150731;第46卷(第14期);全文 * |
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