CN106950318A - A kind of method that ASE HPLC methods determine baicalin in Scutellaria baicalensis Georgi content - Google Patents
A kind of method that ASE HPLC methods determine baicalin in Scutellaria baicalensis Georgi content Download PDFInfo
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- CN106950318A CN106950318A CN201710259567.4A CN201710259567A CN106950318A CN 106950318 A CN106950318 A CN 106950318A CN 201710259567 A CN201710259567 A CN 201710259567A CN 106950318 A CN106950318 A CN 106950318A
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a kind of method that ASE HPLC methods determine baicalin in Scutellaria baicalensis Georgi content, this method extraction effect is good and time-consuming short, reproducible, and accuracy of detection is high.Belong to field of chemical detection.This method extracts Baical Skullcap Root using ASE methods, and collects alcohol extraction liquid；The content of scutelloside in alcohol extraction liquid is determined using HPLC methods.The present invention can be extracted and assay instead of official method to the scutelloside in the root of large-flowered skullcap.
Description
Technical field
The invention belongs to field of chemical detection, especially a kind of ASE-HPLC methods determine the side of baicalin in Scutellaria baicalensis Georgi content
Method.
Background technology
The method for preparing need testing solution described in version official method in 2010 is：Powder about 0.3g in this product is taken, it is accurate
It is weighed, plus 70% ethanol 40ml, it is heated to reflux 3 hours, lets cool, filters, filtrate is put in 100ml measuring bottles, with a small amount of 70% ethanol
In gradation washing container and residue, the washing lotion filter same measuring bottle of people, plus 70% ethanol shakes up to scale.Precision measures 1ml, puts
In 10ml measuring bottles, plus methanol is to scale, shakes up, and produces.
This method return time is long, and requires higher to the professional standards of technical staff.
The content of the invention
For above-mentioned deficiency, the present invention is intended to provide a kind of method that ASE-HPLC methods determine baicalin in Scutellaria baicalensis Georgi content,
This method extraction effect is good and time-consuming short, reproducible, and accuracy of detection is high.
In order to realize above-mentioned technique effect, the technical scheme that the present invention is provided is such：A kind of ASE-HPLC methods are determined
The method of baicalin in Scutellaria baicalensis Georgi content, comprises the steps successively：
Step 1：Baical Skullcap Root is extracted using ASE methods, and collects alcohol extraction liquid；
Step 2：The content of scutelloside in alcohol extraction liquid is determined using HPLC methods.
Preferably, described step 1 includes following sub-steps successively：
Step S1：By root of large-flowered skullcap sample comminution, sieving takes 0.3g to be mixed with 1g quartz sands；
Step S2：The mixture of gained is loaded on and is placed with the ASE abstraction pools of filter membrane, plus quartz sand is extremely parallel with pond mouthful；
Step S3：Extracted with 70% ethanol, after terminating, extract is settled to 50ml with 70% ethanol, centrifuged, take supernatant
Liquid, is produced.
Preferably, the extraction parameters described in step S3 are：Pressure is 1500psi, and temperature is 120 DEG C, and extraction time is
5min, cycle-index is 3 times, and flush volume is 100%, and purge time is 100s.
Preferably, the volume of the ASE abstraction pools described in step S2 is 10ml.
Preferably, the parameter of noncentricity described in step S3 is：Centrifugal speed is 15000r/min, and centrifugation time is 3min.
Preferably, the detection parameter of the HPLC methods described in step 1 is：Chromatographic column is Thermo Syncronis C18；Post
Temperature is 40 DEG C；Flow velocity is 0.5mL/min；Mobile phase is the phosphoric acid of methanol -0.1%；Detection wavelength is 280nm.
Preferably, described chromatographic column specification is：3*100mm, 3 μm.
Preferably, described mobile phase is that volume ratio is equal to 47：53 phosphoric acid of methanol -0.1%.
The present invention is compared with conventional method, with advantages below：
The present invention is once investigated to the solvent that Accelerate solvent extraction is used,《Chinese Pharmacopoeia》Use 70% alcohol reflux 3
Hour.This experiment has carried out solvent investigation from methanol, ethanol, 70% ethanol as Extraction solvent, as a result shows methanol and second
Content of baicalin and version in 2015 after alcohol extracting《Chinese Pharmacopoeia》Content declines, and reason is probably that the polarity of scutelloside is big, therefore is adopted
With Extraction solvent 70% ethanol consistent with pharmacopeia.
The molten boiling point of 70% ethanol is higher, and Accelerate solvent extraction (hereinafter referred to as ASE) extracts principle：A:Improve temperature reduction
The viscosity of solvent, reduce solvent enter sample matrices prevention, add solvent enter sample matrices diffusion, reduction solvent and
Surface tension between sample matrices, makes solvent dissolve the capacity increase of determinand；B:The boiling point of extraction liquids is with pressure rise
And improve, so that solvent remains at liquid at high temperature under high pressure, (liquid is to the solvability of solute much larger than gas to molten
The solvability of matter).Still select to use 120 DEG C, extract 5min, extract 3 times, you can reach it is close with pharmacopeia content, more
Many extraction time, temperature, number of times have had no significant effect to result.It is final to determine that optimum extraction process is combined as Extracting temperature
120 DEG C, extraction time 5min, extraction time 3 times.
Brief description of the drawings
Fig. 1 is the linear regression graph carried out with the concentration (mg/ml) of scutelloside reference substance-peak area；
Fig. 2 is the chromatogram of scutelloside reference substance；
Fig. 3 is the chromatogram using the need testing solution obtained by official method extraction；
Fig. 4 is the chromatogram using the need testing solution obtained by the extraction of ASE methods.
Embodiment
With reference to embodiment, the claim to the present invention is described further, but is not constituted to the present invention
Any limitation, it is any the present invention claims in made limited number of time modification, still the present invention right
In claimed scope.
Embodiment 1
1st, instrument and equipment and reagent
1.1 instruments：
Electronic analytical balance (XA205DU), ASE350 Accelerate solvent extractions instrument (DIONEX companies of the U.S.), Thermo
U3000 UHPLC liquid chromatographs.
1.2 reagents：
Water：Meet one-level water as defined in GB/T 6682；
Ethanol (CH4O)：Chromatographically pure (phase chromatography-use)；
Quartz sand：Particle diameter 2mm.
2nd, method
The preparation of 2.1 need testing solutions
2.1.1 version official method in 2010：
Powder about 0.3g in this product is taken, it is accurately weighed, plus 70% ethanol 40ml, it is heated to reflux 3 hours, lets cool, filters, filtrate
Put in 100ml measuring bottles, with a small amount of 70% ethanol gradation washing container and residue, washing lotion is filtered in the same measuring bottle of people, plus 70% ethanol
To scale, shake up.Precision measures 1ml, puts in 10ml measuring bottles, plus methanol is to scale, shakes up, and produces.
2.1.2 Accelerate solvent extraction method (ASE) prepares test sample method：
The size-reduced machine of sample is crushed, and crosses No. three sieves, accurately weighed 0.3g is well mixed with 1g quartz sands, stand-by, is moved into
Put filter membrane well in advanceAppropriate amount of quartz sand is added in 10ml abstraction pools, gently shaking is allowed to Chi Kou in same water
On horizontal line, tighten and covered on abstraction pool.After extraction terminates, extract is shifted in 50ml volumetric flasks, is diluted to 70% ethanol
Scale, centrifuges 3min under 15000r/min, takes supernatant, is determined into LC.
The preparation of 2.2 reference substance solutions：
Take scutelloside reference substance appropriate, it is accurately weighed, plus solution of every 1ml containing 0.3mg is made in methanol, produces.
2.3 ASE extraction conditions：
Pressure is 1500psi, and temperature is 120 DEG C, and extraction time is 5min, and cycle-index is 3 times, and flush volume is
100%, purge time is 100s.
2.4 chromatographic conditions and system suitability：
A) instrument：Double ternary liquid phase (U-3000) No.5121 of Thermo；
B) chromatographic column：Thermo Syncronis C18,3*100mm, 3 μm；
C) column temperature：40℃；
D) flow velocity：0.5mL/min；
E) mobile phase：：The phosphoric acid of methanol -0.1% (47:53)；
F) Detection wavelength：280nm；
Using octadecylsilane chemically bonded silica as filler；Number of theoretical plate is calculated by psoralen peak should be not less than 2500.
2.5 determination methods：
Determined according to high performance liquid chromatography (general rule 0512)；
It is accurate respectively to draw reference substance solution and each 2 μ l of need testing solution, liquid chromatograph is injected, determines, produces.
The requirement of 2.6 standard limited values：
This product is calculated by dry product, containing scutelloside (C21H18O11) 9.0% must not be less than.
2.7 calculate (external standard method)：
C in formulaR- reference substance solution concentration, unit is micro- gram per liter (mg/L)；
AXThe peak area of-test sample；
AR- reference substance peak area.
Note：
It should be dismantled and cleaned out using preceding extraction bottom of pond portion, otherwise easily cause pressure instability；
Otherwise the filter paper of abstraction pool bottom should cause seepage in sealing ring；
Abstraction pool fills elastic moderate during sample, and too loose to be easily caused extract solution excessive；
Check whether gas cylinder air pressure reaches 1Mpa before start；
Using being cleaned out after terminating, abstraction pool will in time dry and (get rusty easily).
3rd, result
3.1 linear relationships
Take scutelloside reference substance appropriate, it is accurately weighed, put in brown measuring bottle, plus every 1ml is made containing scutelloside in 70% ethanol
0.2852mg solution, then the accurate solution 0.5 μ l, 0.8 μ l, 1 μ l, 2 μ l, the 5 μ l of drawing are determined into LC respectively, and according to
The above method is determined, the results detailed in Table 1.
Linear regression is carried out with concentration (mg/ml)-peak area, regression equation is tried to achieve：Y=6021.6316x-32.5278,
R2=1.0000.Scutelloside is in good linear relationship in the range of 0.1426~1.426mg/ml, refers to Fig. 1.
The Linear Experiment result of table 1
3.2 reappearances are tested
Take the sample (lot number of identical lot number：1510077) 0.3g, it is totally 4 parts, accurately weighed, extract and supply by ASE extracting methods
Test sample solution, sample size is 1 μ L, and with above-mentioned chromatographic condition parallel test, the content for measuring scutelloside in sample is shown in Table 2, RSD
For 0.01%, as a result show that ASE extracting methods repeatability is good.
The reappearance experimental result of table 2
3.3 sample size measurement results (3 batches)
The sample size measurement result of table 3
3.4 chromatographic determinations
Fig. 2 is the chromatogram of scutelloside reference substance；Fig. 3 is the chromatogram using the need testing solution obtained by official method extraction
Figure；Fig. 4 is the chromatogram using the need testing solution obtained by the extraction of ASE methods.
4th, discuss：
The selection of 4.1 ASE Extraction solvents：
The present invention is once investigated to the solvent that Accelerate solvent extraction is used,《Chinese Pharmacopoeia》Use 70% alcohol reflux 3
Hour.This experiment has carried out solvent investigation from methanol, ethanol, 70% ethanol as Extraction solvent, as a result shows methanol and second
Content of baicalin and version in 2015 after alcohol extracting《Chinese Pharmacopoeia》Content declines, and reason is probably that the polarity of scutelloside is big, therefore is adopted
With Extraction solvent 70% ethanol consistent with pharmacopeia.
The optimization of 4.2 ASE extraction conditions
The molten boiling point of 70% ethanol is higher, and Accelerate solvent extraction (hereinafter referred to as ASE) extracts principle：A:Improve temperature reduction
The viscosity of solvent, reduce solvent enter sample matrices prevention, add solvent enter sample matrices diffusion, reduction solvent and
Surface tension between sample matrices, makes solvent dissolve the capacity increase of determinand；B:The boiling point of extraction liquids is with pressure rise
And improve, so that solvent remains at liquid at high temperature under high pressure, (liquid is to the solvability of solute much larger than gas to molten
The solvability of matter).Still select to use 120 DEG C, extract 5min, extract 3 times, you can reach it is close with pharmacopeia content, more
Many extraction time, temperature, number of times have had no significant effect to result.It is final to determine that optimum extraction process is combined as Extracting temperature
120 DEG C, extraction time 5min, extraction time 3 times.
Above-described is only presently preferred embodiments of the present invention, all timess done in the range of the spirit and principles in the present invention
What modification, equivalent and improvement etc., in the hungry protection domain that should be included in the present invention.
Claims (8)
1. a kind of method that ASE-HPLC methods determine baicalin in Scutellaria baicalensis Georgi content, it is characterised in that comprise the steps successively：
Step 1：Baical Skullcap Root is extracted using ASE methods, and collects alcohol extraction liquid；
Step 2：The content of scutelloside in alcohol extraction liquid is determined using HPLC methods.
2. the method that a kind of ASE-HPLC methods according to claim 1 determine baicalin in Scutellaria baicalensis Georgi content, it is characterised in that
Described step 1 includes following sub-steps successively：
Step S1：By root of large-flowered skullcap sample comminution, sieving takes 0.3g to be mixed with 1g quartz sands；
Step S2：The mixture of gained is loaded on and is placed with the ASE abstraction pools of filter membrane, plus quartz sand is extremely parallel with pond mouthful；
Step S3：Extracted with 70% ethanol, after terminating, extract be settled to 50ml with 70% ethanol, centrifuged, take supernatant,
Produce.
3. the method that a kind of ASE-HPLC methods according to claim 2 determine baicalin in Scutellaria baicalensis Georgi content, it is characterised in that
Extraction parameters described in step S3 are：Pressure is 1500psi, and temperature is 120 DEG C, and extraction time is 5min, and cycle-index is 3
Secondary, flush volume is 100%, and purge time is 100s.
4. the method that a kind of ASE-HPLC methods according to claim 2 determine baicalin in Scutellaria baicalensis Georgi content, it is characterised in that
The volume of ASE abstraction pools described in step S2 is 10ml.
5. the method that a kind of ASE-HPLC methods according to claim 2 determine baicalin in Scutellaria baicalensis Georgi content, it is characterised in that
Parameter of noncentricity described in step S3 is：Centrifugal speed is 15000r/min, and centrifugation time is 3min.
6. the method that a kind of ASE-HPLC methods according to claim 1 determine baicalin in Scutellaria baicalensis Georgi content, it is characterised in that
The detection parameter of HPLC methods described in step 1 is：Chromatographic column is Thermo Syncronis C18；Column temperature is 40 DEG C；Flow velocity is
0.5mL/min；Mobile phase is the phosphoric acid of methanol -0.1%；Detection wavelength is 280nm.
7. the method that a kind of ASE-HPLC methods according to claim 6 determine baicalin in Scutellaria baicalensis Georgi content, it is characterised in that
Described chromatographic column specification is：3*100mm, 3 μm.
8. the method that a kind of ASE-HPLC methods according to claim 6 determine baicalin in Scutellaria baicalensis Georgi content, it is characterised in that
Described mobile phase is that volume ratio is equal to 47：53 phosphoric acid of methanol -0.1%.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102225958A (en) * | 2011-05-27 | 2011-10-26 | 浙江大学 | Scutellarin purifying method |
| CN104710392A (en) * | 2015-03-31 | 2015-06-17 | 山西大学 | Method for preparing baicalein by using baicalin |
| CN104829666A (en) * | 2015-05-20 | 2015-08-12 | 山西大学 | Method for preparing high purity baicalin from radix scutellariae |
| CN104910225A (en) * | 2015-05-12 | 2015-09-16 | 广西壮族自治区梧州食品药品检验所 | Method for extracting baicalin from radix scutellariae |
| CN105353053A (en) * | 2015-12-11 | 2016-02-24 | 河北中医学院 | Content determination method for scutellarin and scutellarein in sculellaria barbata medicinal material and formula granule of sculellaria barbata medicinal material |
| CN105784885A (en) * | 2016-04-27 | 2016-07-20 | 广西壮族自治区梧州食品药品检验所 | Method for extracting baicalin and baicalein in scutellaria baicalensis |
| CN105929073A (en) * | 2016-04-27 | 2016-09-07 | 广西壮族自治区梧州食品药品检验所 | Method for simultaneously detecting baicalin and baicalein in Radix Scutellariae |
-
2017
- 2017-04-20 CN CN201710259567.4A patent/CN106950318A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102225958A (en) * | 2011-05-27 | 2011-10-26 | 浙江大学 | Scutellarin purifying method |
| CN104710392A (en) * | 2015-03-31 | 2015-06-17 | 山西大学 | Method for preparing baicalein by using baicalin |
| CN104910225A (en) * | 2015-05-12 | 2015-09-16 | 广西壮族自治区梧州食品药品检验所 | Method for extracting baicalin from radix scutellariae |
| CN104829666A (en) * | 2015-05-20 | 2015-08-12 | 山西大学 | Method for preparing high purity baicalin from radix scutellariae |
| CN105353053A (en) * | 2015-12-11 | 2016-02-24 | 河北中医学院 | Content determination method for scutellarin and scutellarein in sculellaria barbata medicinal material and formula granule of sculellaria barbata medicinal material |
| CN105784885A (en) * | 2016-04-27 | 2016-07-20 | 广西壮族自治区梧州食品药品检验所 | Method for extracting baicalin and baicalein in scutellaria baicalensis |
| CN105929073A (en) * | 2016-04-27 | 2016-09-07 | 广西壮族自治区梧州食品药品检验所 | Method for simultaneously detecting baicalin and baicalein in Radix Scutellariae |
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Application publication date: 20170714 |