CN108169351A - A kind of method that ASE-HPLC methods measure calycosin glucoside content in Radix Astragali - Google Patents
A kind of method that ASE-HPLC methods measure calycosin glucoside content in Radix Astragali Download PDFInfo
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- CN108169351A CN108169351A CN201711364425.0A CN201711364425A CN108169351A CN 108169351 A CN108169351 A CN 108169351A CN 201711364425 A CN201711364425 A CN 201711364425A CN 108169351 A CN108169351 A CN 108169351A
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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
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a kind of method that ASE HPLC methods measure calycosin glucoside content in Radix Astragali, this method takes less when extracting, and accuracy of detection is high, belongs to field of chemical detection.This method extracts Milkvetch Root using ASE methods, and collects methanol extraction liquid；The content of calycosin glucoside in methanol extraction liquid is measured using HPLC methods.The present invention calycosin glucoside in Radix Astragali can be extracted instead of official method and content detection.
Description
Technical field
The invention belongs to field of chemical detection, especially a kind of ASE-HPLC methods measure calycosin glucose in Radix Astragali
The method of glycosides content.
Background technology
《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：The preparation of test solution takes this product powder (to cross four
Number sieve) about 1g, it is accurately weighed, it puts in round-bottomed flask, precision adds in methanol 50ml, and weighed weight is heated to reflux 4 hours, lets cool,
Weighed weight again is supplied the weight of less loss with methanol, is shaken up, filtration, and precision measures subsequent filtrate 25ml, and recycling design is residual to dry
Slag adds methanol to dissolve, and is transferred in 5ml measuring bottles, adds methanol to scale, shake up to get.
This method return time is longer, and higher to the professional standards of technical staff.
Invention content
Against the above deficiency, the present invention is intended to provide a kind of ASE-HPLC methods measure calycosin glucoside in Radix Astragali
The method of content, this method take less when extracting, and accuracy of detection is high.
In order to realize above-mentioned technique effect, technical solution provided by the invention is such：A kind of ASE-HPLC methods measure
The method of calycosin glucoside content, includes the following steps successively in Radix Astragali：
Step 1：Milkvetch Root is extracted, and collect methanol extraction liquid using ASE methods；
Step 2：The content of calycosin glucoside in methanol extraction liquid is measured using HPLC methods.
Preferably, the step 1 includes following sub-steps：
Step S1：By Radix Astragali sample comminution, No. three sieves are crossed, 0.25g is taken to be mixed with 1g diatomite；
Step S2：By the mixture obtained by step S1 loaded on being placed in the ASE abstraction pools of filter membrane, add diatomite to and Chi Kou
It is parallel；
Step S3：With methanol extraction, after, after being evaporated, again with methanol is settled to 10ml, filtering.
Preferably, the extraction parameters described in step S3 are：Pressure is 1500psi, and temperature is 110 DEG C, time 5min, secondary
Number is 3 times, flush volume 60%, purge time 60%.
Preferably, the detection parameters of the HPLC methods described in step 2 are instrument：Chromatographic column is Thermo Syncronis
Dim.AQ；Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is ethyl alcohol-Acetic Acid-Water；Detection wave is 283nm.
Preferably, the specification of the chromatographic column is 1.7 μm, 50 × 2.1mm.
Preferably, the mobile phase is equal to 35 for volume ratio:4:65 methanol-acetic acid-water.
Preferably, the volume of the ASE abstraction pools is 10ml.
Preferably, filter membrane used in the filtration step described in step S3 is 0.22 μm.
Compared with conventional method, tool has the advantage that the present invention：
The present invention once investigated the solvent that Accelerate solvent extraction uses, and has paper to mention and extracts hair using 50% ethyl alcohol
Stamen isoflavones glucoside relative amount is higher, and impurity is less, therefore to methanol and 50% ethyl alcohol is used to carry out solvent investigation, it ties
Fruit shows that methanol and 50% ethyl alcohol extraction calycosin glucoside content are consistent, and impurity peaks are essentially identical, thus use with
The consistent Extraction solvent of pharmacopeia.
It is molten to accelerating to have investigated 3 factors using 3 factor 3 horizontal quadrature experimental program L9 (33) experiment arrangement by the present invention
The influence of calycosin glucoside process in agent extraction Radix Astragali.And finally determine the fast solvent extraction of best glucoside
Taking technique.
Specific embodiment
With reference to embodiment, claims of the present invention is described in further detail, but do not formed
Any limitation of the invention, any limited number of time modification made in the claims of the present invention, still in this hair
In bright claims.
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
1.2 reagent：
Water：Meet level-one water as defined in GB/T 6682；
Methanol (CH4O)：Chromatographically pure (phase chromatography-use)；
Acetic acid (CH3COOH)：It analyzes pure.
1.3 material
Radix Astragali (place of production Inner Mongol, lot number 1511100；Place of production Gansu, lot number 15081901；The place of production Inner Mongol, lot number
150718) Wuzhou or Yulin medicinal material market are purchased from, is differentiated through Guangxi Wuzhou food and medicine inspection institute deputy director pharmacist of traditional Chinese medicine's Huang Heng.
Reference substance calycosin glucoside is provided by Products in China calibrating research institute, lot number：111920-
201505。
2nd, method
The preparation of 2.1 reference substance solutions：Take calycosin glucoside reference substance appropriate, it is accurately weighed, add methanol system
Into every 1ml containing 50 μ g solution to get.
The preparation of 2.2 test solutions
《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：The preparation of test solution takes this product powder (to cross four
Number sieve) about 1g, it is accurately weighed, it puts in round-bottomed flask, precision adds in methanol 50ml, and weighed weight is heated to reflux 4 hours, lets cool,
Weighed weight again is supplied the weight of less loss with methanol, is shaken up, filtration, and precision measures subsequent filtrate 25ml, and recycling design is residual to dry
Slag adds methanol to dissolve, and is transferred in 5ml measuring bottles, adds methanol to scale, shake up to get.
Accelerate solvent extraction method (ASE) prepares test sample method：Sample is crushed through pulverizer, crosses No. four sieves, and about 1g is accurate
It is weighed, it is uniformly mixed with 1g diatomite, for use, is moved into the ASE 10ml abstraction pools for putting filter membrane well in advance and adds in right amount
Diatomite, gently shaking are allowed to Chi Kou in the same horizontal line, tighten abstraction pool upper cover.After extraction, extract liquor is turned
It moves in evaporating dish and dissolves and be settled to 10mL volumetric flasks after being evaporated with methanol, solution enters HPLC surveys after crossing 0.22 micron membrane filter
It is fixed.
2.3ASE extraction conditions
Pressure is 1500psi, and temperature is 110 DEG C, time 5min, and number is 3 times, flush volume 60%, during purging
Between be 60%.
2.4 chromatographic condition and system suitability
A) instrument：1290 high performance liquid chromatographs of Agilent；
B) chromatographic column：Thermo Syncronis Dim. (mm) AQ, 1.7 μm, 50 × 2.1mm；
C) column temperature：40℃；
D) flow velocity：0.3mL/min；
E) mobile phase：：Methanol-acetic acid-water (35:4:65)；
F) Detection wavelength：283nm；
Using octadecylsilane chemically bonded silica as filler；Number of theoretical plate should not by the calculating of calycosin glucoside peak
Less than 3000.
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 10 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, and glucoside containing calycosin (C22H22O10) must not be less than 0.020%.
2.7 calculate (external standard method)
C in formulaR- reference substance solution concentration, unit are micro- gram per liter (mg/L)；
AXThe 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 booting；
It is cleaned out after use, abstraction pool will be dried and (be got rusty easily) in time.
3rd, result
3.1 linear relationship
Take calycosin glucoside reference substance solution (0.06358mg/ml), respectively it is accurate draw 0.1 μ l of the solution,
0.3 μ l, 0.5 μ l, 1.0 μ l, 1.2 μ l enter LC measure, and are measured according to the above method.With concentration (ng)-peak area into line
Property return, acquire regression equation：Y=11.334x+22.62, R2=0.9914, calycosin glucoside 6.358~
76.296ng it is in good linear relationship in the range of.
The Linear Experiment result of 1 calycosin glucoside of table
3.2 repetitive test
Take the sample (lot number of identical lot number：1511100) 1.0g, it is totally 5 parts, accurately weighed, it extracts and supplies by ASE extracting methods
Test sample solution, sample size are 1 μ L, with above-mentioned chromatographic condition parallel test, measure sample result and are shown in Table 1, RSD 1.9%.
2 repeated experiment result of table
| Lot number | Repeatability 1 | Repeatability 2 | Repeatability 3 | Repeatability 4 | Repeatability 5 | RSD% |
| 1511100 | 0.0285 | 0.0289 | 0.0277 | 0.0285 | 0.0291 | 1.9% |
3.3 precision and stability test
Calycosin glucoside reference substance solution (0.06358mg/ml) is taken, continuous sample introduction 6 times records peak area,
Peak area RSD is 2.3%, shows that instrument precision is good.
Same test solution is taken, is placed at room temperature, is measured in accordance with the law respectively at 0,2,4,8,12h.As a result Astragaloside IV peak
The RSD of area is 2.7%, shows that test solution is stablized in 12h.
3.4 sample difference instrument extract result and with official method results contrast (3 batches)
Table 3
Table 4
4th, it discusses：
The selection of 4.1ASE Extraction solvents：
Once the solvent that Accelerate solvent extraction uses is investigated in an experiment, has paper to mention and is extracted using 50% ethyl alcohol
Calycosin glucoside relative amount is higher, and impurity is less, therefore to methanol and 50% ethyl alcohol is used to carry out solvent investigation,
As a result it shows that methanol is consistent with 50% ethyl alcohol extraction calycosin glucoside content, and impurity peaks are essentially identical, therefore uses
The Extraction solvent consistent with pharmacopeia.
The optimization of 4.2ASE extraction conditions
On above-mentioned experiment basis, we use 3 factor 3 horizontal quadrature experimental program L9 (33) experiment arrangement, have investigated 3
Influence of a factor to calycosin glucoside process in accelerated solvent extraction Radix Astragali.This experiment determines extraction temperature, quiet
State extraction time, cycle-index are investigation factor (being shown in Table 5).
It determines that l extracts the best Accelerate solvent extraction technique of calycosin glucoside from Radix Astragali, the results are shown in Table 6.
5 factor level of table
6 orthogonal experiment range analysis result of table
Extracting temperature influences the content of calycosin glucoside maximum it can be seen from range analysis, secondly
Extraction time and extraction time.Finally determining optimum extraction process is combined as 110 DEG C, extraction time 5min of Extracting temperature, extracts
Number 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 modifications, equivalent substitutions and improvements etc., should be included within the scope of the present invention.
Claims (8)
1. a kind of method that ASE-HPLC methods measure calycosin glucoside content in Radix Astragali, which is characterized in that wrap successively
Include following step：
Step 1：Milkvetch Root is extracted, and collect methanol extraction liquid using ASE methods；
Step 2：The content of calycosin glucoside in methanol extraction liquid is measured using HPLC methods.
2. a kind of ASE-HPLC methods according to claim 1 measure the side of calycosin glucoside content in Radix Astragali
Method, which is characterized in that the step 1 includes following sub-steps：
Step S1：By Radix Astragali sample comminution, No. three sieves are crossed, 0.25g is taken to be mixed with 1g diatomite；
Step S2：By the mixture obtained by step S1 loaded on being placed in the ASE abstraction pools of filter membrane, diatomite is added extremely to be put down with Chi Kou
Row；
Step S3：With methanol extraction, after, after being evaporated, again with methanol is settled to 10ml, filtering.
3. a kind of ASE-HPLC methods according to claim 2 measure the side of calycosin glucoside content in Radix Astragali
Method, which is characterized in that the extraction parameters described in step S3 are：Pressure is 1500psi, and temperature is 110 DEG C, time 5min, secondary
Number is 3 times, flush volume 60%, purge time 60%.
4. a kind of ASE-HPLC methods according to claim 1 measure the side of calycosin glucoside content in Radix Astragali
Method, which is characterized in that the detection parameters of the HPLC methods described in step 2 are instrument：Chromatographic column is Thermo Syncronis
Dim.AQ；Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is ethyl alcohol-Acetic Acid-Water；Detection wave is 283nm.
5. a kind of ASE-HPLC methods according to claim 4 measure the side of calycosin glucoside content in Radix Astragali
Method, which is characterized in that the specification of the chromatographic column is 1.7 μm, 50 × 2.1mm.
6. a kind of ASE-HPLC methods according to claim 4 measure the side of calycosin glucoside content in Radix Astragali
Method, which is characterized in that the mobile phase is equal to 35 for volume ratio:4:65 methanol-acetic acid-water.
7. a kind of ASE-HPLC methods according to claim 2 measure the side of calycosin glucoside content in Radix Astragali
Method, which is characterized in that the volume of the ASE abstraction pools is 10ml.
8. a kind of ASE-HPLC methods according to claim 2 measure the side of calycosin glucoside content in Radix Astragali
Method, which is characterized in that filter membrane used in the filtration step described in step S3 is 0.22 μm.
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