CN107688067A - The content assaying method of TONGXIAO BIYAN PIAN - Google Patents
The content assaying method of TONGXIAO BIYAN PIAN Download PDFInfo
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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
Abstract
The present invention relates to the content assaying method of TONGXIAO BIYAN PIAN, more particularly to the content assaying method of active ingredient macrotin glycosides in TONGXIAO BIYAN PIAN, 5 O methyl visamminols glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin and Isomperatorin, including the preparation of need testing solution, the preparation of reference substance solution, UPLC methods measure.Using content assaying method provided by the invention, in measure, detection baseline is steady, separating degree is good, accuracy in detection is high, stability is good；The assay time greatly is shortened, improves detection efficiency steadily, toxic chemical dosage is greatly decreased, more economical environmental protection；And multi-target ingredient detection is carried out simultaneously, a kind of rapidly and efficiently feasible detection method is provided for the foundation in TONGXIAO BIYAN PIAN liquid-phase fingerprint storehouse, easily can be the study on the stability of TONGXIAO BIYAN PIAN, there is provided method is investigated in a kind of quantitative comparison of more comprehensively stable effective ingredients.
Description
Technical field
The present invention relates to the assay of Chinese patent drug, more particularly, to active ingredient macrotin glycosides in TONGXIAO BIYAN PIAN,
5-O- methyl visamminols glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, the assay side of Imperatorin and Isomperatorin
Method.
Background technology
TONGXIAO BIYAN PIAN is by the Six-element Chinese medicine such as stir-baked FRUCCTUS XANTHII, windproof, the Radix Astragali, the root of Dahurain angelica, the flower bud of lily magnolia, rhizoma atractylodis macrocephalae, peppermint, warp
Pure Chinese medicine compound preparation made of extraction, with the effect of solid table of scattered wind, facilitaing lung is sensible.Accumulate lung, exterior deficiency institute for wind-heat
Weight, thin nasal discharge or turbid tears, forehead headache during the nasal obstruction of cause；The illness such as rhinitis chronic, allergic rhinitis, nasosinusitis.It is sensible
There is principle active component in rhinitis tablet：Macrotin glycosides, 5-O- methyl visamminols glycosides, calycosin glucoside, Ou Qian
Hu Su, Isomperatorin and magnelin etc..
But the important active ingredient of these in sensible rhinitis does not have and realizes more comprehensive geology by the detection of science
Amount detection.
The primary standard of TONGXIAO BIYAN PIAN record in《Pharmacopoeia of People's Republic of China》2015 editions one, in standard only to Europe before
Hu Su has carried out content detection.Report has the discriminating of active ingredient and content assaying method in TONGXIAO BIYAN PIAN in existing literature,
But the discriminating being related to and assay method, most trivial operations, time-consuming, just in TONGXIAO BIYAN PIAN simply medicinal material or one into
Point carry out quantitative control, it is impossible to qualitative and quantitative control comprehensively is carried out to TONGXIAO BIYAN PIAN product, and can not be realized while more
Component target detects, so as to be unable to the quality of science control TONGXIAO BIYAN PIAN product.
Existing literature《HPLC-MS methods determine the content of 6 compositions in anti-root of Dahurian angelica nasopharynx particle simultaneously》,《Pharmaceutical Analysis magazine》
ChinJPharmAnal2014.34 (5), Liu Linggai；Zhi Xuran etc., disclose one kind HPLC-MS methods while determine anti-root of Dahurian angelica nose
The method for swallowing 6 component contents in particle, this method determine a kind of content of 6 kinds of compositions in Chinese medicine preparation simultaneously, but should
Method is the method for LC-MS, in practical operation, that is, needs to start the liquid phase using mobile phase elution, needs startup to adopt again
Need to complete within 30 minutes by the use of high alcohol nitrogen as the mass spectrum of atomization gas, detection time, efficiency is low, and organic solvent and high pure nitrogen disappear
Consumption is big, and testing cost is high, and LC-MS easily produces reappearance and the degree of accuracy that matrix effect influences to detect.
Existing literature《HPLC methods determine the content of 5 kinds of active ingredient in four seasons common cold piece simultaneously》,《Chinese pharmacists》, 2015
Year volume 18 the 3rd phase China Pharmacist 2015, Vol.18No.3, Zheng Zhaoxian；Zhao Dantong etc., disclose a kind of use
The method that HPLC methods determine 5 kinds of active constituent contents in four seasons common cold piece simultaneously, this method determine a kind of Chinese medicine preparation simultaneously
In 5 kinds of compositions content, but this method is in actual mechanical process, and appearance time length, detection time is up to 50 minutes, efficiency
Low, organic solvent consumption is big.And only two kinds of compositions are identical with the present invention.
Existing literature《RP-HPLC methods determine 5 kinds of compositions in TONGXIAO BIYAN PIAN simultaneously》,《Northwest University's journal (natural science
Version)》, in April, 2015, the 2nd phase of volume 45, Apr., 2015, Vo1.45, No.2, Zhang Qian, Wang Shixiang etc., disclose a kind of use
The method that RP-HPLC methods determine 5 kinds of component contents in TONGXIAO BIYAN PIAN simultaneously, this method detection time are up to 80 minutes, efficiency
It is too low, and five kinds of compositions are all from root of Dahurain angelica medicinal material simply, and to the active ingredient of other medicinal materials in TONGXIAO BIYAN PIAN without fixed
Amount control, therefore the optimum detection method of non-TONGXIAO BIYAN PIAN content detection.
《Pharmacopoeia of People's Republic of China》Containing for root of Dahurain angelica Imperatorin in TONGXIAO BIYAN PIAN is disclosed in 2015 editions one
Quantity measuring method, it is as follows：" chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With first
Alcohol-water (54:46) it is mobile phase；Detection wavelength is 248nm.Number of theoretical plate is calculated by Imperatorin peak should be not less than 5000. pairs
Prepared according to product solution：Take Imperatorin reference substance appropriate, it is accurately weighed, add methanol that the solution that every 1mL contains 15 μ g is made, produce.
The preparation of need testing solution：This product 20 is taken, sugar coated tablet removes sugar-coat, accurately weighed, finely ground, takes about 2g, accurately weighed, puts tool
To fill in conical flask, precision adds methanol 25mL, weighed weight, is ultrasonically treated (power 250w, frequency 50Hz) 30min, lets cool, then
Weighed weight, the weight of less loss is supplied with methanol, is shaken up, filtered, take subsequent filtrate, produce.Determination method：It is accurate respectively to draw control
Product solution and each 10 μ L of need testing solution, liquid chromatograph is injected, measure, is produced." this method chromatographic condition and side of the present invention
Method difference is obvious, surveys the Imperatorin that composition is only limitted in the root of Dahurain angelica.
《Pharmacopoeia of People's Republic of China》Tongqiao Biyan capsules detection method of content is disclosed in 2015 editions one, detection refers to
It is the Imperatorin (the same TONGXIAO BIYAN PIAN of method) and Astragaloside IV in Radix Astragali in the root of Dahurain angelica to mark composition.The assay of Astragaloside IV
Method is as follows：" chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With acetonitrile-water
(32:68) it is mobile phase；EISD detects.Number of theoretical plate is calculated by Astragaloside IV should be not less than 3000. controls
It is prepared by product solution：Take Astragaloside IV reference substance appropriate, it is accurately weighed, add methanol that solution of every 1mL containing 0.5mg is made, produce.For
The preparation of test sample solution：This product 30 is taken, pours out content, it is accurately weighed, it is finely ground, about 4g is taken, it is accurately weighed, put soxhlet extraction
In device, methanol 50mL is added, is heated to reflux 4 hours, extract solution recycling design is simultaneously concentrated to dryness, and residue adds water 10mL, and low-grade fever makes
Dissolving, with water saturated n-butanol shaking extraction 4 times, each 40mL, merge n-butanol liquid, fully wash 2 times with ammonia solution, often
Secondary 40mL, ammoniacal liquor is discarded, for n-butanol liquid recycling design to doing, residue adds water 5mL to make dissolving, lets cool, and crosses D101 type macroreticular resins
Post (internal diameter 1.5cm, pillar height 12cm), eluted with water 50mL, discard aqueous, then eluted with 40% ethanol 30mL, discarded and wash
De- liquid, eluted after with 70% ethanol 80mL, collect eluent, recycling design is dissolved with methanol to doing and is transferred to 5mL volumetric flasks
In, add methanol to shake up, produce to scale.Determination method：Precision draws μ L of reference substance solution 5,15 μ L and each 10 μ of need testing solution
L, liquid chromatograph is injected, measure, is calculated, produced with external standard two-point method logarithmic equation." this method test sample preparation method answers
Miscellaneous, time-consuming.Although it is controlled two content detection indexs, two kinds of different detection methods, cost are used
Height, time-consuming, and efficiency is low.
Above method can not all detect 6 kinds of active ingredients in TONGXIAO BIYAN PIAN simultaneously.And these method detection times
Long, efficiency is low, and organic solvent consumption is big, the polyphamaceutic composition of TONGXIAO BIYAN PIAN is determined while can not be quick and precisely
Amount analysis.
In order to preferably detect the content of active ingredient in TONGXIAO BIYAN PIAN, the quality of TONGXIAO BIYAN PIAN is lifted, research carries
For one kind is easy to operate, specificity is strong, separating degree is good, stability is good, high-efficiency environment friendly, and can carry out multi-target ingredient control simultaneously
TONGXIAO BIYAN PIAN detection method of content, it is imperative.
The content of the invention
The purpose of the present invention is the defects of overcoming prior art, there is provided one kind is directed to active ingredient rattletop in TONGXIAO BIYAN PIAN
Plain glycosides, 5-O- methyl visamminols glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin and containing for Isomperatorin measure
Determine method, content assaying method provided by the invention, in detection, detection baseline is steady, separating degree is good, accuracy in detection is high,
Stability is good；The assay time greatly is shortened, improves detection efficiency steadily, toxic chemical dosage significantly subtracts
It is few, more economical environmental protection；And the detection of multi-target ingredient is carried out simultaneously, it is the foundation in the liquid-phase fingerprint storehouse of TONGXIAO BIYAN PIAN
A kind of rapidly and efficiently feasible detection method is provided, easily can be the study on the stability of TONGXIAO BIYAN PIAN, there is provided a kind of more comprehensive
Method is investigated in the quantitative comparison of stable effective ingredients.
Specifically, the present invention provides active ingredient macrotin glycosides, 5-O- methyl Wei Sia meter in a kind of TONGXIAO BIYAN PIAN
Alcohol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, the content assaying method of Imperatorin and Isomperatorin, methods described include pair
Active ingredient macrotin glycosides in preparation, 5-O- methyl visamminols glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin and
The assay of Isomperatorin；The assay comprises the following steps：The preparation of need testing solution, the system of reference substance solution
Standby, UPLC methods measure.
Preferably, the chromatographic condition of the UPLC methods measure is：Waters BEH C18 posts；Mobile phase A is acetonitrile, flowing
Phase B is the acetic acid that volume fraction is 0.4 ‰~0.8 ‰, gradient elution method；0.2~0.5mL/min of flow velocity, Detection wavelength
254nm, 35 DEG C of column temperature.
In the chromatographic condition, in order to realize more excellent separating effect, the Waters BEH C18 posts it is interior
Footpath size is 50mm × 2.1mm, 1.7 μm.
As a kind of preferred scheme, the gradient elution method is：0~2.6min, 10%~20% → 60%~80%
A；2.6~3.5min, 60%~80% → 80%~100%A；3.5~4.0min, 80%~100%A；4.0~4.5min,
80%~100% → 10%~20%A；4.5~7min, 10%~20%A.
In order to improve separating degree, and the content for detecting 6 kinds of different active ingredients simultaneously is realized, it is further preferred that institute
Stating gradient elution method is：0~2.6min, 18% → 70%A；2.6~3.5min, 70% → 95%A；3.5~4.0min,
95%A；4.0~4.5min, 95% → 18%A；4.5~7min, 18%A.
Preferably, the preparation method of the need testing solution is：TONGXIAO BIYAN PIAN 1~3g of powder is taken, precision is weighed, put
In 100~200mL conical flask with stopper or volumetric flask, precision adds 30~60mL of methanol of volume fraction 60~80%, weighs,
100W, 25kHz 30~60min of ultrasonic extraction, let cool, are re-weighed, and supplement weightlessness with the methanol of volume fraction 60~80%, use is micro-
Hole membrane filtration, subsequent filtrate is taken as need testing solution.
It is highly preferred that the preparation method of the need testing solution is：TONGXIAO BIYAN PIAN powder 1.0g is taken, precision is weighed, put
In 100mL conical flask with stopper or volumetric flask, precision adds the methanol 50mL of volume fraction 80%, weighs, 100W, 25kHz ultrasounds carry
45min is taken, is let cool, is re-weighed, weightlessness is supplied with the methanol of volume fraction 80%, with 0.22 μm of filtering with microporous membrane, takes subsequent filtrate
As need testing solution.
Reference substance solution of the present invention includes macrotin glycosides reference substance solution, 5-O- methyl visamminol glycosides reference substances
Solution, calycosin glucoside reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance solution and wood
Blue fat element reference substance solution.
The preparation method of the macrotin glycosides reference substance solution is：Macrotin glycosides reference substance is taken, adds methanol to be dissolved to concentration
It is standby for 45~55 μ g/mL；The preparation method of the 5-O- methyl visamminol glycosides reference substance solution is：Take 5-O- methyl
Visamminol glycosides reference substance, methanol is added to be dissolved to concentration as 75~85 μ g/mL, it is standby；The calycosin glucoside
The preparation method of reference substance solution is：Calycosin glucoside reference substance is taken, it is 45~55 μ g/ to add methanol to be dissolved to concentration
ML, it is standby；The preparation method of the Imperatorin reference substance solution is：Imperatorin reference substance is taken, adds methanol to be dissolved to concentration
It is standby for 55~65 μ g/mL；The preparation method of the Isomperatorin reference substance solution is：Isomperatorin reference substance is taken, is added
Methanol is dissolved to concentration as 180~220 μ g/mL, standby；The preparation method of the magnelin reference substance solution is：Take lily magnolia
Fat element reference substance, methanol is added to be dissolved to concentration as 45~55 μ g/mL, it is standby；By the macrotin glycosides reference substance solution of preparation, 5-O-
It is methyl visamminol glycosides reference substance solution, calycosin glucoside reference substance solution, Imperatorin reference substance solution, different
Imperatorin reference substance solution and magnelin reference substance solution, by 2~6:5~9:2~8:2~8:15~25:2~8 ratios
It is deployed into the mixed reference substance solution of 6 kinds of compositions.
Preferably, the preparation method of the macrotin glycosides reference substance solution is：Macrotin glycosides reference substance is taken, adds methanol to dissolve
It is 50 μ g/mL to concentration, it is standby；The preparation method of the 5-O- methyl visamminol glycosides reference substance solution is：Take 5-O- first
Base visamminol glycosides reference substance, methanol is added to be dissolved to concentration as 80 μ g/mL, it is standby；The calycosin glucoside pair
Preparation method according to product solution is：Calycosin glucoside reference substance is taken, adds methanol to be dissolved to concentration as 50 μ g/mL, it is standby
With；The preparation method of the Imperatorin reference substance solution is：Imperatorin reference substance is taken, it is 60 μ to add methanol to be dissolved to concentration
G/mL, it is standby；The preparation method of the Isomperatorin reference substance solution is：Isomperatorin reference substance is taken, adds methanol to dissolve
It is 200 μ g/mL to concentration, it is standby；The preparation method of the magnelin reference substance solution is：Magnelin reference substance is taken, is added
Methanol is dissolved to concentration as 50 μ g/mL, standby；By the macrotin glycosides reference substance solution of preparation, 5-O- methyl visamminol glycosides
Reference substance solution, calycosin glucoside reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance are molten
Liquid and magnelin reference substance solution, by 5:8:5:6:20:5 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions.
As a preferred embodiment of the present invention, active ingredient macrotin glycosides, 5-O- methyl dimension in the TONGXIAO BIYAN PIAN
This ammiol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, the content assaying method of Imperatorin and Isomperatorin include following step
Suddenly：
(1) preparation of need testing solution：TONGXIAO BIYAN PIAN 1~3g of powder is taken, precision weighs, and puts 100~200mL tool plug
In triangular flask or volumetric flask, precision adds 30~60mL of methanol of volume fraction 60~80%, weighs, 100W, 25kHz ultrasounds carry
30~60min is taken, is let cool, is re-weighed, weightlessness is supplemented with the methanol of volume fraction 60~80%, with filtering with microporous membrane, takes continuous
Filtrate is as need testing solution；
(2) preparation of reference substance solution：Take macrotin glycosides reference substance, 5-O- methyl visamminol glycosides reference substance, Mao Rui
Isoflavones glucoside reference substance, Imperatorin reference substance, Isomperatorin reference substance and magnelin reference substance, add methanol molten
Solution, it is 45~55 μ g/mL macrotin glycosides reference substance solution, 75~85 μ g/mL 5-O- methyl Wei Sia that concentration, which is respectively prepared,
Rice alcohol glycosides reference substance solution, 45~55 μ g/mL calycosin glucoside reference substance solution, 55~65 μ g/mL Europe before
The magnelin control of plain reference substance solution, 180~220 μ g/mL Isomperatorin reference substance solution and 45~55 μ g/mL recklessly
Product solution, it is standby；
By the macrotin glycosides reference substance solution of preparation, 5-O- methyl visamminol glycosides reference substance solution, calycosin
Glucoside reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance solution and magnelin reference substance are molten
Liquid, by 2~6:5~9:2~8:2~8:15~25:2~8 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions, standby；
(3) UPLC methods determine：It is accurate respectively to draw need testing solution and each 1~5 μ L of mixed reference substance solution, inject superelevation
Effect liquid phase chromatogram instrument, determine, calculate, produce；
The chromatographic condition of UPLC methods measure is：Waters BEH C18 posts, internal diameter are 50mm × 2.1mm, 1.7 μm；
Mobile phase A is acetonitrile, and Mobile phase B is the acetic acid that volume fraction is 0.4 ‰~0.8 ‰；Gradient elution：0~2.6min, 10%~
20% → 60%~80%A；2.6~3.5min, 60%~80% → 80%~100%A；3.5~4.0min, 80%~
100%A；4.0~4.5min, 80%~100% → 10%~20%A；4.5~7min, 10%~20%A；Flow velocity 0.2~
0.5mL/min, Detection wavelength 254nm, 35 DEG C of column temperature.
It is further preferred that active ingredient macrotin glycosides, 5-O- methyl visamminols glycosides, hair in the TONGXIAO BIYAN PIAN
Stamen isoflavone aglycone, magnelin, the content assaying method of Imperatorin and Isomperatorin comprise the following steps：
(1) preparation of need testing solution：Take TONGXIAO BIYAN PIAN powder 1.0g, precision weighs, put 100mL conical flask with stopper or
In volumetric flask, precision adds the methanol 50mL of volume fraction 80%, weighs, 100W, 25kHz ultrasonic extraction 45min, lets cool, then claim
Weight, supplies weightlessness with the methanol of volume fraction 80%, with 0.22 μm of filtering with microporous membrane, takes subsequent filtrate as need testing solution；
(2) preparation of reference substance solution：Take macrotin glycosides reference substance, 5-O- methyl visamminol glycosides reference substance, Mao Rui
Isoflavones glucoside reference substance, Imperatorin reference substance, Isomperatorin reference substance and magnelin reference substance, add methanol molten
Solution, it is 50 μ g/mL macrotin glycosides reference substance solution, 80 μ g/mL 5-O- methyl visamminol glycosides pair that concentration, which is respectively prepared,
According to product solution, 50 μ g/mL calycosin glucoside reference substance solution, 60 μ g/mL Imperatorin reference substance solution,
The magnelin reference substance solution of 200 μ g/mL Isomperatorin reference substance solution and 50 μ g/mL, it is standby；
By the macrotin glycosides reference substance solution of preparation, 5-O- methyl visamminol glycosides reference substance solution, calycosin
Glucoside reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance solution and magnelin reference substance are molten
Liquid, by 5:8:5:6:20:5 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions, standby；
(3) UPLC methods determine：It is accurate respectively to draw need testing solution and each 1 μ L of mixed reference substance solution, inject ultra high efficiency
Liquid chromatograph, determine, calculate, produce；
The chromatographic condition of UPLC methods measure is：Waters BEH C18 posts, internal diameter are 50mm × 2.1mm, 1.7 μm；
Mobile phase A is acetonitrile, and Mobile phase B is the acetic acid that volume fraction is 0.4 ‰~0.8 ‰；Gradient elution：0~2.6min, 18% →
70%A；2.6~3.5min, 70% → 95%A；3.5~4.0min, 95%A；4.0~4.5min, 95% → 18%A；4.5~
7min, 18%A；0.2~0.5mL/min of flow velocity, Detection wavelength 254nm, 35 DEG C of column temperature.
Through actually detected, the content of macrotin glycosides is that 300~500 μ g/g, 5-O- methyl tie up this in the TONGXIAO BIYAN PIAN
The content of ammiol glycosides is 700~900 μ g/g, and Calycosin-7-O-BETA-D-glucoside content is 150~300 μ g/g, and magnelin content is 300
~500 μ g/g, Imperatorin content are 800~1000 μ g/g, and Isomperatorin content is 700~900 μ g/g.
Preferably, the content of macrotin glycosides is 413 μ g/g, 5-O- methyl visamminol glycosides in the TONGXIAO BIYAN PIAN
Content is 830 μ g/g, and Calycosin-7-O-BETA-D-glucoside content is 254 μ g/g, and magnelin content is 485 μ g/g, and Imperatorin content is
955 μ g/g, Isomperatorin content are 873 μ g/g.
The present invention realizes following beneficial effect：
1st, the method for ultra high efficiency is established first, while detects 6 kinds of active ingredients in TONGXIAO BIYAN PIAN, is realized and is referred to more
The detection of composition is marked, the foundation for the liquid-phase fingerprint storehouse of TONGXIAO BIYAN PIAN provides a kind of rapidly and efficiently feasible detection side
Method, easily can be the study on the stability of TONGXIAO BIYAN PIAN, there is provided a kind of quantitative comparison of more comprehensively stable effective ingredients is investigated
Method.
2nd, the gradient elution method of suitable TONGXIAO BIYAN PIAN effective substance is accurately summarized, detection time is by common liquid
50~80min of phase shortens to 7min, greatly shortens the assay time, improves detection efficiency steadily, meanwhile, greatly
The usage amount (3.5mL is tapered to by 50~80mL) for reducing mobile phase, mobile phase more containing the toxic solvent such as methanol or acetonitrile,
Dosage is reduced, significantly reduces the pollution to environment, it is more environmentally-friendly, also reduce testing cost.
3 while it have detected six kinds of most representational active ingredient rattletops of four main Chinese medicinal materials in TONGXIAO BIYAN PIAN
Plain glycosides, 5-O- methyl visamminols glycosides, Calycosin-7-O-BETA-D-glucoside, lily magnolia fat, Imperatorin and Isomperatorin.
4th, the method for the invention, detection baseline is steady, separating degree is high, accuracy in detection is high, stability is good.
Brief description of the drawings
Fig. 1 is the UPLC chromatograms of mixed reference substance solution in embodiment 1；
Fig. 2 is the UPLC chromatograms of need testing solution in embodiment 1.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.Unless stated otherwise, the raw material used in the present embodiment and equipment are the art routinely original purchased in market
Material and equipment.
The instrument and reagent being related in following embodiment include：Ultra performance liquid chromatography：Waters ACQuity
UPLC (Water companies of the U.S.), KQ5200DE types numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)；Electronics day
Flat (Beijing Sai Duolisi balances Co., Ltd)；Ultrapure water machine (ABW-1001-U type Ai Kepu).Reference substance (is purchased from Chinese food
Drug assay research institute)：Macrotin glycosides (lot number：111710-200501)；5-O- methyl visamminol glycosides (lot numbers：
111523-201509)；Calycosin glucoside (lot number：111920-201505)；Magnelin (lot number：110882-
201206)；Imperatorin (lot number：110826-201415)；Isomperatorin (lot number：110827-201410).
Embodiment 1 detects active ingredient macrotin glycosides in TONGXIAO BIYAN PIAN, 5-O- methyl Wei Sia meter in accordance with the following methods
Alcohol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin, the content of Isomperatorin：
(1) preparation of need testing solution：TONGXIAO BIYAN PIAN powder 1.0g is taken, precision weighs, and puts 100mL conical flask with stopper
In, precision adds the methanol 50mL of volume fraction 80%, weighs, 100W, 25kHz ultrasonic extraction 45min, lets cool, be re-weighed, use body
The methanol of fraction 80% supplies weightlessness, with 0.22 μm of filtering with microporous membrane, takes subsequent filtrate as need testing solution；
(2) preparation of reference substance solution：Take macrotin glycosides reference substance, 5-O- methyl visamminol glycosides reference substance, Mao Rui
Isoflavones glucoside reference substance, Imperatorin reference substance, Isomperatorin reference substance and magnelin reference substance, add methanol molten
Solution, it is 50 μ g/mL macrotin glycosides reference substance solution, 80 μ g/mL 5-O- methyl visamminol glycosides pair that concentration, which is respectively prepared,
According to product solution, 50 μ g/mL calycosin glucoside reference substance solution, 60 μ g/mL Imperatorin reference substance solution,
The magnelin reference substance solution of 200 μ g/mL Isomperatorin reference substance solution and 50 μ g/mL, it is standby；
By the macrotin glycosides reference substance solution of preparation, 5-O- methyl visamminol glycosides reference substance solution, calycosin
Glucoside reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance solution and magnelin reference substance are molten
Liquid, by 5:8:5:6:20:5 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions, standby；
(3) UPLC methods determine：It is accurate respectively to draw need testing solution and each 1 μ L of mixed reference substance solution, inject ultra high efficiency
Liquid chromatograph, determine, calculate, produce；
The chromatographic condition of UPLC methods measure is：Waters BEH C18 posts, internal diameter are 50mm × 2.1mm, 1.7 μm；
Mobile phase A is acetonitrile, and Mobile phase B is the acetic acid that volume fraction is 0.6 ‰；Gradient elution：0~2.6min, 18% → 70%A；
2.6~3.5min, 70% → 95%A；3.5~4.0min, 95%A；4.0~4.5min, 95% → 18%A；4.5~7min,
18%A；Flow velocity 0.5mL/min, Detection wavelength 254nm, 35 DEG C of column temperature.
Testing result shows that detection baseline is steady, and each target component separating degree is good in sample, and sample appearance time exists
4~8 minutes, detection time can be completed within 8 minutes.
Embodiment 2 detects active ingredient macrotin glycosides in TONGXIAO BIYAN PIAN, 5-O- methyl Wei Sia meter in accordance with the following methods
Alcohol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin, the content of Isomperatorin：
(1) preparation of need testing solution：TONGXIAO BIYAN PIAN powder 3.0g is taken, precision weighs, and puts in 200mL volumetric flasks, essence
The close addition methanol 30mL of volume fraction 60%, weighs, 100W, 25kHz ultrasonic extraction 30min, lets cool, be re-weighed, use volume integral
Several 60% methanol supply weightlessness, with 0.22 μm of filtering with microporous membrane, take subsequent filtrate as need testing solution；
(2) preparation of reference substance solution：Take macrotin glycosides reference substance, 5-O- methyl visamminol glycosides reference substance, Mao Rui
Isoflavones glucoside reference substance, Imperatorin reference substance, Isomperatorin reference substance and magnelin reference substance, add methanol molten
Solution, it is 45 μ g/mL macrotin glycosides reference substance solution, 75 μ g/mL 5-O- methyl visamminol glycosides pair that concentration, which is respectively prepared,
According to product solution, 45 μ g/mL calycosin glucoside reference substance solution, 55 μ g/mL Imperatorin reference substance solution,
The magnelin reference substance solution of 180 μ g/mL Isomperatorin reference substance solution and 45 μ g/mL, it is standby；
By the macrotin glycosides reference substance solution of preparation, 5-O- methyl visamminol glycosides reference substance solution, calycosin
Glucoside reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance solution and magnelin reference substance are molten
Liquid, by 2:5:2:2:15:2 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions, standby；
(3) UPLC methods determine：It is accurate respectively to draw need testing solution and each 5 μ L of mixed reference substance solution, inject ultra high efficiency
Liquid chromatograph, determine, calculate, produce；
The chromatographic condition of UPLC methods measure is：Waters BEH C18 posts, internal diameter are 50mm × 2.1mm, 1.7 μm；
Mobile phase A is acetonitrile, and Mobile phase B is the acetic acid that volume fraction is 0.4 ‰；Gradient elution：0~2.6min, 10% → 60%A；
2.6~3.5min, 60% → 80%A；3.5~4.0min, 80%A；4.0~4.5min, 80% → 10%A；4.5~7min,
10%A；Flow velocity 0.2mL/min, Detection wavelength 254nm, 35 DEG C of column temperature.
Testing result shows that detection baseline is steady, and each target component separating degree is good in sample, and effect is slightly worse than embodiment
1, at 4~8 minutes, detection time can be completed sample appearance time within 8 minutes.
Embodiment 3 detects active ingredient macrotin glycosides in TONGXIAO BIYAN PIAN, 5-O- methyl Wei Sia meter in accordance with the following methods
Alcohol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin, the content of Isomperatorin：
(1) preparation of need testing solution：TONGXIAO BIYAN PIAN powder 2.0g is taken, precision weighs, and puts 150mL conical flask with stopper
In, precision adds the methanol 60mL of volume fraction 70%, weighs, 100W, 25kHz ultrasonic extraction 60min, lets cool, be re-weighed, use body
The methanol of fraction 70% supplies weightlessness, with 0.22 μm of filtering with microporous membrane, takes subsequent filtrate as need testing solution；
(2) preparation of reference substance solution：Take macrotin glycosides reference substance, 5-O- methyl visamminol glycosides reference substance, Mao Rui
Isoflavones glucoside reference substance, Imperatorin reference substance, Isomperatorin reference substance and magnelin reference substance, add methanol molten
Solution, it is 55 μ g/mL macrotin glycosides reference substance solution, 85 μ g/mL 5-O- methyl visamminol glycosides pair that concentration, which is respectively prepared,
According to product solution, 55 μ g/mL calycosin glucoside reference substance solution, 65 μ g/mL Imperatorin reference substance solution,
The magnelin reference substance solution of 220 μ g/mL Isomperatorin reference substance solution and 55 μ g/mL, it is standby；
By the macrotin glycosides reference substance solution of preparation, 5-O- methyl visamminol glycosides reference substance solution, calycosin
Glucoside reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance solution and magnelin reference substance are molten
Liquid, by 6:9:8:8:25:8 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions, standby；
(3) UPLC methods determine：It is accurate respectively to draw need testing solution and each 2 μ L of mixed reference substance solution, inject ultra high efficiency
Liquid chromatograph, determine, calculate, produce；
The chromatographic condition of UPLC methods measure is：Waters BEHC18 posts, internal diameter are 50mm × 2.1mm, 1.7 μm；Stream
Dynamic phase A is acetonitrile, and Mobile phase B is the acetic acid that volume fraction is 0.8 ‰；Gradient elution：0~2.6min, 20% → 80%A；2.6
~3.5min, 80% → 100%A；3.5~4.0min, 100%A；4.0~4.5min, 100% → 20%A；4.5~7min,
20%A；Flow velocity 0.3mL/min, Detection wavelength 254nm, 35 DEG C of column temperature.
Testing result shows that detection baseline is steady, and each target component separating degree is good in sample, and effect is slightly worse than embodiment
1, at 4~8 minutes, detection time can be completed sample appearance time within 8 minutes.
Embodiment 4 detects active ingredient macrotin glycosides in TONGXIAO BIYAN PIAN, 5-O- methyl Wei Sia meter in accordance with the following methods
Alcohol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin, the content of Isomperatorin：
(1) preparation of need testing solution：TONGXIAO BIYAN PIAN powder 2.0g is taken, precision weighs, and puts in 200mL volumetric flasks, essence
The close addition methanol 50mL of volume fraction 75%, weighs, 100W, 25kHz ultrasonic extraction 40min, lets cool, be re-weighed, use volume integral
Several 75% methanol supply weightlessness, with 0.22 μm of filtering with microporous membrane, take subsequent filtrate as need testing solution；
(2) preparation of reference substance solution：With embodiment 1；
(3) UPLC methods determine：With embodiment 1；
The chromatographic condition of the UPLC methods measure：With embodiment 1；
Testing result shows that detection baseline is steady, and each target component separating degree is good in sample, and effect and embodiment 1 are poor
Seldom, at 4~8 minutes, detection time can be completed sample appearance time within 8 minutes.
Embodiment 5 detects active ingredient macrotin glycosides in TONGXIAO BIYAN PIAN, 5-O- methyl Wei Sia meter in accordance with the following methods
Alcohol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin, the content of Isomperatorin：
(1) preparation of need testing solution：With embodiment 1；
(2) preparation of reference substance solution：With embodiment 1；
(3) UPLC methods determine：With embodiment 1；
The chromatographic condition of UPLC methods measure is：Waters BEH C18 posts, internal diameter are 50mm × 2.1mm, 1.7 μm；
Mobile phase A is acetonitrile, and Mobile phase B is the acetic acid that volume fraction is 0.2 ‰；Gradient elution：0~2.6min, 25%~→ 55%
A；2.6~3.5min, 55% → 75%A；3.5~4.0min, 75%A；4.0~4.5min, 75% → 25%A；4.5~
7min, 25%A；Flow velocity 0.5mL/min, Detection wavelength 254nm, 35 DEG C of column temperature.
Testing result shows that detection baseline is steady, and each target component separating degree is preferable in sample, and effect is slightly worse than embodiment
1~embodiment 4, at 4~10 minutes, detection time can be completed sample appearance time within 10 minutes.
Comparative example 1 carries out assay to the active ingredient in TONGXIAO BIYAN PIAN in accordance with the following methods：
(1) preparation of need testing solution：With the need testing solution preparation method of embodiment 1；
(2) preparation of reference substance solution：With the reference substance solution preparation method of embodiment 1；
(3) assay method：It is accurate respectively to draw need testing solution and each 20 μ L of mixed reference substance solution, inject liquid chromatogram
Instrument, determine, calculate, produce；
The chromatographic condition of the assay method is：Dikma Diamonsil C18 chromatographic columns (4.6mm × 150mm, 5m),
Mobile phase A is Chromatographic Pure Methanol, and Mobile phase B is the water containing 0.05% formic acid, gradient elution (0~6min, 25%A → 75%A；6
~10min, 75%A → 95%A；10~15min, 95%A), be with mobile phase primary condition balance 6min, flow velocity before sample introduction
0.8mL/min. column temperatures are room temperature, and sample size is 20 μ L.
As a result show, detected using this comparative example method, spectrogram baseline is unstable, appearance time 10 minutes it
Afterwards, detection time needs 15~30 minutes, it is impossible to 5-O- methyl visamminol glycosides is detected, and between target component and impurity
It is not completely separated, assay is unstable.
Comparative example 2 carries out assay to the active ingredient in TONGXIAO BIYAN PIAN in accordance with the following methods：
(1) preparation of need testing solution：With the need testing solution preparation method of embodiment 1；
(2) preparation of reference substance solution：With the reference substance solution preparation method of embodiment 1；
(3) assay method：It is accurate respectively to draw above-mentioned need testing solution, each 10 μ L of reference substance solution, inject liquid chromatogram
Instrument, measure, is produced；
The chromatographic condition of the assay method is：Aglient ZORBAX SB-Cl8 chromatographic columns (250mm × 4.6mm, 5 μ
m)；Mobile phase A is acetonitrile, and B is 0.1% aqueous formic acid, gradient elution (0~35min, 10%A → 35%A；35~
40min, 35%A → 90%A；40~45min, 90%A → l0%A；45~50min, 10%A；Flow velocity：1.0mL/min；Post
Temperature：30℃；Detection wavelength：283nm；Sample size：10μL.
As a result show, detected using this comparative example method, spectrogram baseline is unstable, appearance time 20 minutes it
Afterwards, detection time needs 40~50 minutes, it is impossible to detects Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin, different Imperatoria ostruthium
Element, it is impossible to accurately 6 kinds of target components of measure, do not reach the requirement of quality assurance.Comparative example 3 is in accordance with the following methods to sensible nose
Active ingredient in scorching piece carries out assay：
(1) preparation of need testing solution：With the need testing solution preparation method of embodiment 1；
(2) preparation of reference substance solution：With the reference substance solution preparation method of embodiment 1；
(3) assay method：It is accurate respectively to draw above-mentioned need testing solution, reference substance solution, each 50 μ L of negative control solution,
Liquid chromatograph is injected, measure, is produced；
The chromatographic condition of the assay method is：Agilent TC-C18 chromatographic columns (4.6mm × 250mm, 55 μm), flowing
Phase A is 0.2% formic acid water, and B is acetonitrile, gradient elution (0~42min, 22%B；43~73min, 50%B)；Detection wavelength is
254nm；Volume flow 1.0mL/min；30 DEG C of column temperature；The μ L of sample size 50.
As a result show, detected using this comparative example method, spectrogram baseline is unstable, appearance time 20 minutes it
Afterwards, detection time needs 70~80 minutes, it is impossible to detects macrotin glycosides, 5-O- methyl visamminol glycosides, calycosin
Glycosides, magnelin, it is impossible to accurately 6 kinds of target components of measure, do not reach the requirement of quality assurance.
Comparative example 4
Compared with Example 1, difference is, in chromatographic condition, uses《Chinese Pharmacopoeia 2015 editions》It is first page 1458, public
Chromatographic condition in the TONGXIAO BIYAN PIAN content assaying method opened carries out the assay of TONGXIAO BIYAN PIAN.
As a result show, detected using this comparative example method, for appearance time after 22 minutes, detection time needs 35
Minute, the detection method, it is only capable of detecting Imperatorin, it is impossible to while multi-target ingredient is detected, do not reach multi-flavor medicinal material
Multi-target ingredient quantitatively controls requirement.
Comparative example 5
Compared with Example 1, difference is, in chromatographic condition, uses《Chinese Pharmacopoeia 2015 editions》It is first page 1459, public
Chromatographic condition in the Tongqiao Biyan capsules content assaying method opened carries out the assay of Tongqiao Biyan capsules.
As a result show, detected using this comparative example method, Imperatorin and Astragaloside IV appearance time are respectively 22
Minute, detection time was respectively necessary for 30 minutes and 35 minutes with after 20 minutes, and was to be tried respectively by two kinds of different confessions
Product preparation method and chromatographic test strip part are realized.By two kinds of detection methods, it is only capable of detecting Imperatorin and Radix Astragali first respectively
Glycosides, it is impossible to while detect multi-target ingredient, it is difficult to reach multicomponent quantitative control and require.
The linear relationship investigation of embodiment 6, precision test, replica test, stability test, recovery test and sample
Assay：
According to the method processing need testing solution of embodiment 1, reference substance solution, the same embodiment of chromatographic condition, detection method
1, tested as follows：
(1) linear relationship is investigated：The accurate lower mixed reference substance solution 0.5 of extraction embodiment 1 respectively, 1,2,4,8,12,
16 μ L sample introductions determine.With peak area (Y) for ordinate, sample size (X, μ g) is abscissa, draws standard curve, obtains macrotin
Glycosides, 5-O- methyl visamminols glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, the regression equation of Imperatorin and Isomperatorin
Respectively：Y=3763.02X-304.96 γ=0.9999 (n=6)；Y=2018.12X+22575.6 γ=0.9999 (n=
6)；Y=603.33X-8962.55 γ=0.999 (n=6)；Y=2329.46X+68544.21 γ=0.9998 (n=6)；Y=
2023.62X+41224.11 γ=0.9999 (n=6)；Y=700.55X-1007.56 γ=0.9999 (n=6)；The range of linearity
Respectively：0.025~0.8 μ g；0.04~1.28 μ g；0.025~0.8 μ g；0.03~0.96 μ g；0.03~0.96 μ g；0.10
~3.20 μ g.
(2) precision test：The need testing solution prepared respectively under accurate extraction embodiment 1, continuous sample introduction 6 times, survey
Obtain macrotin glycosides, 5-O- methyl visamminols glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin and Isomperatorin
The RSD of integrating peak areas value is respectively 0.5%, 0.4%, 0.9%, 0.8%, 0.3%, 0.3%, shows that instrument precision is good
It is good.
(3) replica test：Same batch sample is taken, is prepared by 1 lower test sample preparation method of embodiment is parallel for examination
6 parts of product, determined by 1 lower chromatographic condition of embodiment, calculate content.Macrotin glycosides, 5-O- methyl Wei Sia meter in 6 parts of test samples
Alcohol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, the average content of Imperatorin and Isomperatorin are respectively 0.37mg/g,
0.72mg/g, 0.31mg/g, 0.37mg/g, 0.79mg/g, 0.42mg/g, its RSD are respectively：1.0%, 1.2%, 1.8%,
1.6%, 0.8%, 0.6%, show repeated good.
(4) stability test：Same batch sample is taken, it is molten to prepare test sample by 1 lower test sample preparation method of embodiment
Liquid, 0,2,4,6,8,10,12, the 24h sample introduction after preparation, measure macrotin glycosides in test sample, 5-O- methyl visamminols glycosides,
Calycosin-7-O-BETA-D-glucoside, magnelin, the RSD of integrating peak areas value of Imperatorin and Isomperatorin are respectively：1.0%,
1.2%, 1.7%, 1.5%, 0.7%, 0.6%, show that need testing solution is stable in 24h.
(5) recovery test：Test sample 0.5g is taken, 6 parts, accurately weighed, precision adds macrotin glycosides, 5-O- methyl respectively
Visamminol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin and Isomperatorin reference substance are appropriate, by embodiment 1
Lower test sample preparation method is prepared into need testing solution, is measured by 1 lower chromatographic condition of embodiment, calculates the rate of recovery, knot
Fruit macrotin glycosides, 5-O- methyl visamminols glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, Imperatorin and Isomperatorin
The rate of recovery (n=9) is respectively 97.5%, 99.3%, 98.0%, 98.3%, 99.2,99.5%, and its RSD is respectively：1.1%,
1.0%, 1.6%, 1.3%, 1.0%, 0.8%.
(6) sample size determines：Three batches of TONGXIAO BIYAN PIANs are taken, by 1 lower section legal system available test sample solution of embodiment, measure
6 kinds of component contents, as a result see the table below shown：
From above experimental result, the present invention replaces traditional HPLC methods to carry out assay using UPLC methods, significantly
Time and solvent are saved, and achieves preferable peak type and theoretical cam curve, target component and separated from impurities, specificity
It is good, and plurality of active ingredients detection can be carried out simultaneously.
This experiment establish quantitative approach, by the measure of multiple batches of sample, result presentation method is simple, specificity is good and
The rate of recovery is high, and feasibility is strong, is the optimum content detection method as TONGXIAO BIYAN PIAN.
The general principle and the advantage of principal character and the present invention of the present invention has been shown and described above.The technology of this area
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, and this is to this
It is it will be apparent that these changes and improvements all fall within the protetion scope of the claimed invention for art personnel.This hair
Bright claimed scope is by appended claims and its equivalent thereof.
Claims (10)
1. the content assaying method of TONGXIAO BIYAN PIAN, it is characterised in that including：To active ingredient macrotin glycosides, 5-O- in preparation
Methyl visamminol glycosides, Calycosin-7-O-BETA-D-glucoside, magnelin, the assay of Imperatorin and Isomperatorin, it is described to contain
Measurement comprises the following steps surely：The preparation of need testing solution, the preparation of reference substance solution, UPLC methods measure；
The chromatographic condition of UPLC methods measure is：Waters BEH C18 posts；Mobile phase A is acetonitrile, and Mobile phase B is volume integral
Count the acetic acid for 0. 4 ‰~0.8 ‰, gradient elution method；0.2~0.5mL/min of flow velocity, Detection wavelength 254nm, column temperature 35
℃。
2. according to the method for claim 1, it is characterised in that the internal diameter size of the Waters BEH C18 posts is 50mm
× 2.1mm, 1.7 μm.
3. according to the method for claim 1, it is characterised in that the gradient elution method is：0~2.6min, 10%~
20% → 60%~80%A；2.6~3.5min, 60%~80% → 80%~100%A；3.5~4.0min, 80%~100%A；4.0~
4.5min, 80%~100% → 10%~20%A；4.5~7min, 10%~20%A;
Preferably, the gradient elution method is：0~2.6min, 18% → 70%A；2.6~3.5min, 70% → 95%A；3.5~
4.0min, 95%A；4.0~4.5min, 95% → 18%A；4.5~7min, 18% A.
4. according to the method for claim 1, it is characterised in that：The preparation method of the need testing solution is：Take sensible nose
1~3g of scorching piece powder, precision weigh, and put in 100~200mL conical flask with stopper or volumetric flask, and precision adds volume fraction 60
~80% 30~60mL of methanol, weighs, 100W, 25kHz 30~60min of ultrasonic extraction, lets cool, is re-weighed, with volume fraction 60
~80% methanol supplement is weightless, with filtering with microporous membrane, takes subsequent filtrate as need testing solution；
Preferably, the preparation method of the need testing solution is：TONGXIAO BIYAN PIAN powder 1.0g is taken, precision weighs, and puts 100mL tools
To fill in triangular flask or volumetric flask, precision adds the methanol 50mL of volume fraction 80%, weighs, 100W, 25kHz ultrasonic extraction 45min,
Let cool, be re-weighed, supply weightlessness with the methanol of volume fraction 80%, with 0.22 μm of filtering with microporous membrane, take subsequent filtrate as test sample
Solution.
5. according to the method for claim 1, it is characterised in that：It is molten that the reference substance solution includes macrotin glycosides reference substance
Liquid, 5-O- methyl visamminol glycosides reference substance solution, calycosin glucoside reference substance solution, Imperatorin reference substance
Solution, Isomperatorin reference substance solution and magnelin reference substance solution.
6. according to the method for claim 5, it is characterised in that：
The preparation method of the macrotin glycosides reference substance solution is：Macrotin glycosides reference substance is taken, adds methanol to be dissolved to concentration as 45
~55 μ g/mL, it is standby；
The preparation method of the 5-O- methyl visamminol glycosides reference substance solution is：5-O- methyl visamminols glycosides is taken to compare
Product, methanol is added to be dissolved to concentration as 75~85 μ g/mL, it is standby；
The preparation method of the calycosin glucoside reference substance solution is：Calycosin glucoside reference substance is taken,
Methanol is added to be dissolved to concentration as 45~55 μ g/mL, it is standby；
The preparation method of the Imperatorin reference substance solution is：Imperatorin reference substance is taken, adds methanol to be dissolved to concentration as 55
~65 μ g/mL, it is standby；
The preparation method of the Isomperatorin reference substance solution is：Isomperatorin reference substance is taken, adds methanol to be dissolved to concentration
It is standby for 180~220 μ g/mL；
The preparation method of the magnelin reference substance solution is：Magnelin reference substance is taken, adds methanol to be dissolved to concentration as 45
~55 μ g/mL, it is standby；
By the reference substance solution macrotin glycosides of preparation, 5-O- methyl visamminols glycosides, calycosin glucoside, Imperatoria ostruthium
Element, Isomperatorin and magnelin, by 2~6:5~9:2~8:2~8:15~25:2~8 ratios are deployed into 6 kinds of compositions
Mixed reference substance solution.
7. the method according to claim 5 or 6, it is characterised in that：
The preparation method of the macrotin glycosides reference substance solution is：Macrotin glycosides reference substance is taken, it is 50 μ to add methanol to be dissolved to concentration
G/mL, it is standby；
The preparation method of the 5-O- methyl visamminol glycosides reference substance solution is：5-O- methyl visamminols glycosides is taken to compare
Product, methanol is added to be dissolved to concentration as 80 μ g/mL, it is standby；
The preparation method of the calycosin glucoside reference substance solution is：Calycosin glucoside reference substance is taken,
Methanol is added to be dissolved to concentration as 50 μ g/mL, it is standby；
The preparation method of the Imperatorin reference substance solution is：Imperatorin reference substance is taken, it is 60 μ to add methanol to be dissolved to concentration
G/mL, it is standby；
The preparation method of the Isomperatorin reference substance solution is：Isomperatorin reference substance is taken, adds methanol to be dissolved to concentration
It is standby for 200 μ g/mL；
The preparation method of the magnelin reference substance solution is：Magnelin reference substance is taken, it is 50 μ to add methanol to be dissolved to concentration
G/mL, it is standby；
By the reference substance solution macrotin glycosides of preparation, 5-O- methyl visamminols glycosides, calycosin glucoside, Imperatoria ostruthium
Element, Isomperatorin and magnelin, by 5:8:5:6:20:5 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions.
8. the content assaying method of TONGXIAO BIYAN PIAN according to claim 1, it is characterised in that：This method includes following step
Suddenly：
（1）The preparation of need testing solution：TONGXIAO BIYAN PIAN 1~3g of powder is taken, precision weighs, and puts 100~200mL tool plug triangle
In bottle or volumetric flask, precision adds 30~60mL of methanol of volume fraction 60~80%, weighs, 100W, 25kHz ultrasonic extractions 30
~60min, lets cool, and is re-weighed, and supplements weightlessness with the methanol of volume fraction 60~80%, with filtering with microporous membrane, takes subsequent filtrate to make
For need testing solution；
（2）The preparation of reference substance solution：Take macrotin glycosides reference substance, 5-O- methyl visamminol glycosides reference substance, Mao Ruiyi yellow
Ketone glucoside reference substance, Imperatorin reference substance, Isomperatorin reference substance and magnelin reference substance, add methanol to dissolve,
It is 45~55 μ g/mL macrotin glycosides reference substance solution, 75~85 μ g/mL 5-O- methyl visamminols that concentration, which is respectively prepared,
Glycosides reference substance solution, 45~55 μ g/mL calycosin glucoside reference substance solution, 55~65 μ g/mL Imperatorin
The magnelin reference substance of reference substance solution, 180~220 μ g/mL Isomperatorin reference substance solution and 45~55 μ g/mL is molten
Liquid, it is standby；
By the macrotin glycosides reference substance solution of preparation, 5-O- methyl visamminol glycosides reference substance solution, calycosin grape
Glucosides reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance solution and magnelin reference substance solution, are pressed
2~6:5~9:2~8:2~8:15~25:2~8 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions, standby；
（3）UPLC methods determine：It is accurate respectively to draw need testing solution and each 1~5 μ L of mixed reference substance solution, inject ultra high efficiency liquid
Chromatography, determine, calculate, produce；
The chromatographic condition of UPLC methods measure is：Waters BEH C18 posts, internal diameter are 50mm × 2.1mm, 1.7 μm；Flowing
Phase A is acetonitrile, and Mobile phase B is the acetic acid that volume fraction is 0. 4 ‰~0.8 ‰；Gradient elution：0~2.6min, 10%~20%
→ 60%~80%A；2.6~3.5min, 60%~80% → 80%~100%A；3.5~4.0min, 80%~100%A；4.0~
4.5min, 80%~100% → 10%~20%A；4.5~7min, 10%~20% A；0.2~0.5mL/min of flow velocity, Detection wavelength
254nm, 35 DEG C of column temperature.
9. the content assaying method of the TONGXIAO BIYAN PIAN according to claim 1 or 8, it is characterised in that：This method include with
Lower step：
（1）The preparation of need testing solution：TONGXIAO BIYAN PIAN powder 1.0g is taken, precision weighs, and puts 100mL conical flask with stopper or capacity
In bottle, precision adds the methanol 50mL of volume fraction 80%, weighs, 100 W, 25 kHz ultrasonic extraction 45min, lets cool, be re-weighed,
Weightlessness is supplied with the methanol of volume fraction 80%, with 0. 22 μm of filtering with microporous membrane, takes subsequent filtrate as need testing solution；
（2）The preparation of reference substance solution：Take macrotin glycosides reference substance, 5-O- methyl visamminol glycosides reference substance, Mao Ruiyi yellow
Ketone glucoside reference substance, Imperatorin reference substance, Isomperatorin reference substance and magnelin reference substance, add methanol to dissolve,
The 5-O- methyl visamminols glycosides control that concentration is 50 μ g/mL macrotin glycosides reference substance solution, 80 μ g/mL is respectively prepared
Product solution, 50 μ g/mL calycosin glucoside reference substance solution, 60 μ g/mL Imperatorin reference substance solution, 200 μ
The magnelin reference substance solution of g/mL Isomperatorin reference substance solution and 50 μ g/mL, it is standby；
By the macrotin glycosides reference substance solution of preparation, 5-O- methyl visamminol glycosides reference substance solution, calycosin grape
Glucosides reference substance solution, Imperatorin reference substance solution, Isomperatorin reference substance solution and magnelin reference substance solution, are pressed
5:8:5:6:20:5 ratios are deployed into the mixed reference substance solution of 6 kinds of compositions, standby；
（3）UPLC methods determine：It is accurate respectively to draw need testing solution and each 1 μ L of mixed reference substance solution, inject ultra high efficiency liquid phase
Chromatograph, determine, calculate, produce；
The chromatographic condition of UPLC methods measure is：Waters BEH C18 posts, internal diameter are 50mm × 2.1mm, 1.7 μm；Flowing
Phase A is acetonitrile, and Mobile phase B is the acetic acid that volume fraction is 0.4 ‰~0.8 ‰；Gradient elution：0~2.6min, 18% → 70%A；
2.6~3.5min, 70% → 95%A；3.5~4.0min, 95%A；4.0~4.5min, 95% → 18% A；4.5~7min, 18%A；
0.2~0.5mL/min of flow velocity, Detection wavelength 254nm, 35 DEG C of column temperature.
10. the content assaying method described in claim any one of 1-9, it is characterised in that macrotin in the TONGXIAO BIYAN PIAN
The content of glycosides is that the content of 300~500 μ g/g, 5-O- methyl visamminol glycosides is 700~900 μ g/g, Calycosin-7-O-BETA-D-glucoside
Content is 150~300 μ g/g, and magnelin content is 300~500 μ g/g, and Imperatorin content is 800~1000 μ g/g, different
Imperatorin content is 700~900 μ g/g；
Preferably, the content of macrotin glycosides is the content of 413 μ g/g, 5-O- methyl visamminol glycosides in the TONGXIAO BIYAN PIAN
For 830 μ g/g, Calycosin-7-O-BETA-D-glucoside content is 254 μ g/g, and magnelin content is 485 μ g/g, and Imperatorin content is 955 μ
G/g, Isomperatorin content are 873 μ g/g.
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