CN108088928A - A kind of method for measuring Content Determination of Indirubin in folium isatidis - Google Patents
A kind of method for measuring Content Determination of Indirubin in folium isatidis Download PDFInfo
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- CN108088928A CN108088928A CN201711368027.6A CN201711368027A CN108088928A CN 108088928 A CN108088928 A CN 108088928A CN 201711368027 A CN201711368027 A CN 201711368027A CN 108088928 A CN108088928 A CN 108088928A
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- folium isatidis
- indirubin
- content determination
- measuring content
- extraction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- 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
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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
- G01N30/14—Preparation by elimination of some components
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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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- 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
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
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Abstract
The invention discloses a kind of methods for measuring Content Determination of Indirubin in folium isatidis, and this method extraction time is short, reproducible, and accuracy of detection is high.Belong to field of chemical detection.Dyers Woad Leaf is extracted with ethyl acetate using ASE methods in this method, and collects extract liquor；The content of indigo red in extract liquor is measured using HPLC methods.The present invention indigo red in folium isatidis can be extracted instead of official method and assay.
Description
Technical field
The invention belongs to field of chemical detection, especially a kind of method for measuring Content Determination of Indirubin in folium isatidis.
Background technology
《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：This product fine powder 0.25g is taken, it is accurately weighed, put Soxhlet
In extractor, add chloroform, when immersion 15 is small, heating and refluxing extraction to extracting liquid colourless.For recycling design to doing, residue adds second
Alcohol makes dissolving and is transferred in 100ml measuring bottles, and ethyl alcohol is added to shake up to scale, filters, take subsequent filtrate to get.
The present invention is more demanding to the professional standards of technical staff, and return time is longer.
The content of the invention
Against the above deficiency, the present invention is intended to provide a kind of method for measuring Content Determination of Indirubin in folium isatidis, this method extraction
Take the time short, it is reproducible, and accuracy of detection is high.
In order to realize above-mentioned technique effect, technical solution provided by the invention is such：Indigo in a kind of measure folium isatidis
The method of rubine content, comprises the following steps successively：
Step 1：Dyers Woad Leaf is extracted with ethyl acetate using ASE methods, and collects extract liquor；
Step 2：The content of indigo red in extract liquor is measured using HPLC methods.
Preferably, the step 1 includes following sub-steps successively：
Step S1：By folium isatidis sample comminution, No. three sieves are crossed, 0.25g is taken to be mixed with 1g quartz sands；
Step S2：By the mixture of gained loaded on being placed in the ASE abstraction pools of filter membrane, add diatomite extremely parallel with pond mouthful；
Step S3：It is extracted with ethyl acetate, after, extract liquor is settled to 50ml with ethyl alcohol.
Preferably, the extraction step parameter described in step S3 is：Extraction temperature is 90 DEG C, extraction time 5min, Xun Huan
Number is 2 times, flush volume 60%, purge time 60s.
Preferably, the detection parameters of the HPLC methods described in step 2 are：Chromatographic column is Thermo Syncronis C18；Column
Temperature is 40 DEG C；Flow velocity is 0.5mL/min；Mobile phase is alcohol-water；Detection wavelength is 289nm.
Preferably, the specification of the chromatographic column is 3 μm, 3 × 100mm.
Preferably, the mobile phase is equal to 75 for volume ratio：25 alcohol-water.
Preferably, the volume of the ASE abstraction pools described in step S2 is 5ml.
The present invention has the following advantages compared with conventional method：
The present invention has investigated ethyl acetate, n-hexane, ethyl acetate：Normal hexane (1:1) as a result, ethyl alcohol represents, ethyl alcohol and
Ethyl acetate extraction efficiency is basically identical, but the impurity peaks of ethyl alcohol extraction are more, therefore this experiment uses ethyl acetate molten for extraction
Agent.
The present invention uses 3 factor, 3 horizontal quadrature experiment investigation, three factors being affected to extraction efficiency：Extraction temperature
Degree, static extracting time, cycle-index by experimental result, with reference to range analysis, determine preferable ASE parameters for extraction temperature eventually
90 DEG C, static extracting time 5min of degree, cycle-index 2 times.
Specific embodiment
With reference to embodiment, the claim of the present invention is described further, but do not formed to the present invention
Any restrictions, it is any made in the claims in the present invention protection domain limited number of time modification, still the present invention right will
It asks in protection domain.
Embodiment 1
1st, instrument and equipment and reagent
1.1 instrument：
Electronic analytical balance (XA205DU), ASE350 Accelerate solvent extractions instrument (DIONEX companies of the U.S.), Thermo
U3000 UHPLC liquid chromatographs, Agilent ultrahigh speed liquid chromatograph
1.2 reagent：Reagent and solution (in addition to especially indicating, this experiment agents useful for same is that analysis is pure)
Water：Meet level-one water as defined in GB/T 6682；
Ethyl alcohol (CH4O)：Chromatographically pure (phase chromatography-use).
2nd, method
The preparation of 2.1 reference substance solutions：
Take indigo red reference substance appropriate, it is accurately weighed, add ethyl alcohol be made every 1ml containing 2 μ g solution to get.
The preparation of 2.2 test solutions
2.2.1《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：
Take this product fine powder 0.25g, it is accurately weighed, put in apparatus,Soxhlet's, add chloroform, impregnate 15 it is small when, heat back
Stream is extracted to extracting liquid colourless.To doing, residue adds ethyl alcohol to make dissolving and is transferred in 100ml measuring bottles recycling design, adds ethyl alcohol extremely
Scale shakes up, filtration, take subsequent filtrate to get.
2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method：
Sample is crushed through pulverizer, crosses No. three sieves, and about 0.25g is accurately weighed, is uniformly mixed with 1g quartz sands, for use, is moved
Enter into the 5ml abstraction pools for putting filter membrane well in advance and add appropriate diatomite, gently shaking is allowed to Chi Kou in same level
On line, abstraction pool upper cover is tightened.After extraction, extract liquor transfer in 50ml volumetric flasks, scale is diluted to ethyl alcohol, is filtered
Cross, take subsequent filtrate to get.
2.3ASE extraction conditions：Extractant is ethyl acetate, and extraction temperature is 90 DEG C, extraction time 5min, Xun Huan
Number is 2 times, flush volume 60%, purge time 60s.
2.4 chromatographic conditions and system suitability
A) instrument：Thermo double ternary liquid phase (U-3000), 1290 ultrahigh speed liquid chromatographs of Agilent；
B) chromatographic column：Thermo Syncronis(C18 3μm 3×100mm)；
C) column temperature：40℃；
D) flow velocity：0.5mL/min；
E) mobile phase：：Alcohol-water (75:25)；
F) Detection wavelength：289nm；
Using octadecylsilane chemically bonded silica as filler；Number of theoretical plate is calculated by indigo red peak should be not less than 4000.
2.5 measuring method
It is measured according to high performance liquid chromatography (general rule 0512)
It is accurate respectively to draw reference substance solution and each 2 μ l of test solution, inject liquid chromatograph, measure to get.
The requirement of 2.6 standard limited values
This product is calculated by dry product, containing indigo red (C16H10N2O2) 0.020% must not be less than.
2.7 calculate (external standard method)
C in formulaR--- reference substance solution concentration, unit are micrograms per millilitre (μ g/mL)；
AX--- the peak area of test sample；
AR--- reference substance peak area.
Pay attention to：
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 leakage in sealing ring；
Abstraction pool fills elastic moderate during sample, and too loose to be easy to cause extracting solution excessive；
Check whether gas cylinder air pressure reaches 1Mpa before start；
It is cleaned out after use, abstraction pool will be dried and (got rusty easily) in time.
3rd, result
3.1 linear relationship
Take indigo red reference substance solution (concentration：0.00175mg/ml), it is accurate respectively to draw 0.5 μ l of the solution, 1 μ l, 2 μ
L, 3 μ l, 5 μ l enter LC measure, and are measured according to the above method, the results detailed in Table 1.
Linear regression is carried out with concentration (μ g)-average peak area, acquires regression equation：Y=12.841x-1.0345, R2=
1.Indigo red is in good linear relationship in the range of 0.875~8.75ng.
1 Linear Experiment result of table
3.2 repeated experiment
Take the sample (lot number of identical lot number：1509003) 0.25g, it is totally 5 parts, accurately weighed, it is extracted by ASE extracting methods
Test solution, sample size are 2 μ l, and with above-mentioned chromatographic condition parallel test, the content for measuring indigo red in sample is shown in Table 2,
RSD is 1.7%, the results showed that ASE extracting methods repeatability is good.
2 repeatability of table realizes result
3.3 precision test
Indigo red reference substance solution (0.00175mg/ml) is taken, continuous sample introduction 6 times records peak area, and peak area RSD is
1.5%, show that instrument precision is good.
3.4 sample difference instruments extract result and with official method results contrast (3 batches)
The different instrument extraction results contrasts of table 3
Table 4ASE extracts results contrast with pharmacopeia
4th, discuss：
The selection of 4.1ASE Extraction solvents：
《Chinese Pharmacopoeia》Using indigo red in chloroform recovery folium isatidis, chloroform toxicity is big, and the physicochemical property of indigo red is
Be dissolved in ethyl acetate, acetone, chloroform, ether, it is not soluble in water, be slightly soluble in ethyl alcohol, this experiment investigated ethyl acetate, n-hexane,
Ethyl acetate：Normal hexane (1:1), ethyl alcohol, as a result such as table 5, ethyl alcohol and ethyl acetate extraction efficiency are basically identical, but ethyl alcohol extracts
Impurity peaks it is more, therefore this experiment uses ethyl acetate as Extraction solvent.
5 Extraction solvent of table investigates result
| Extraction solvent | Measure content (%) |
| Ethyl alcohol | 0.118 |
| Ethyl acetate | 0.111 |
| N-hexane | 0.097 |
| N-hexane：Ethyl acetate (1:1) | 0.085 |
The optimization of 4.2ASE extraction conditions
This experiment uses 3 factor, 3 horizontal quadrature experiment investigation, three factors being affected to extraction efficiency：Extraction temperature
Degree, static extracting time, cycle-index by result of the test, determine preferably ASE parameters, are shown in Table 6, table 7.
6 factor level of table designs table
7 orthogonal experiment range analysis result of table
The static extracting time influences the extraction efficiency of indigo red maximum it can be seen from range analysis, followed by extracts
Temperature and cycle-index.Preferable ASE parameters are finally determined as 90 DEG C of extraction temperature, static extracting time 5min, cycle-index 2
It is secondary.
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 modifications, equivalent substitutions and improvements etc., should be included within the scope of the present invention.
Claims (7)
- A kind of 1. method for measuring Content Determination of Indirubin in folium isatidis, which is characterized in that comprise the following steps successively：Step 1：Dyers Woad Leaf is extracted with ethyl acetate using ASE methods, and collects extract liquor；Step 2：The content of indigo red in extract liquor is measured using HPLC methods.
- A kind of 2. method for measuring Content Determination of Indirubin in folium isatidis according to claim 1, which is characterized in that the step Rapid 1 includes following sub-steps successively：Step S1：By folium isatidis sample comminution, No. three sieves are crossed, 0.25g is taken to be mixed with 1g quartz sands；Step S2：By the mixture of gained loaded on being placed in the ASE abstraction pools of filter membrane, add diatomite extremely parallel with pond mouthful；Step S3：It is extracted with ethyl acetate, after, extract liquor is settled to 50ml with ethyl alcohol.
- A kind of 3. method for measuring Content Determination of Indirubin in folium isatidis according to claim 2, which is characterized in that step S3 institutes The extraction step parameter stated is：Extraction temperature is 90 DEG C, extraction time 5min, and cycle-index is 2 times, and flush volume is 60%, purge time 60s.
- A kind of 4. method for measuring Content Determination of Indirubin in folium isatidis according to claim 1, which is characterized in that step 2 institute The detection parameters for the HPLC methods stated are：Chromatographic column is Thermo Syncronis C18；Column temperature is 40 DEG C；Flow velocity is 0.5mL/ min；Mobile phase is alcohol-water；Detection wavelength is 289nm.
- A kind of 5. method for measuring Content Determination of Indirubin in folium isatidis according to claim 4, which is characterized in that the color The specification for composing column is 3 μm, 3 × 100mm.
- A kind of 6. method for measuring Content Determination of Indirubin in folium isatidis according to claim 4, which is characterized in that the stream It is dynamic to be mutually equal to 75 for volume ratio：25 alcohol-water.
- A kind of 7. method for measuring Content Determination of Indirubin in folium isatidis according to claim 2, which is characterized in that step S2 institutes The volume for the ASE abstraction pools stated is 5ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109916963A (en) * | 2019-04-24 | 2019-06-21 | 东莞维科电池有限公司 | The test method of water content in a kind of slurry |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109916963A (en) * | 2019-04-24 | 2019-06-21 | 东莞维科电池有限公司 | The test method of water content in a kind of slurry |
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Application publication date: 20180529 |