CN106918665A - A kind of method that ASE HPLC methods determine magnelin content in the flower bud of lily magnolia - Google Patents
A kind of method that ASE HPLC methods determine magnelin content in the flower bud of lily magnolia Download PDFInfo
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- CN106918665A CN106918665A CN201710232457.9A CN201710232457A CN106918665A CN 106918665 A CN106918665 A CN 106918665A CN 201710232457 A CN201710232457 A CN 201710232457A CN 106918665 A CN106918665 A CN 106918665A
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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
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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 determine magnelin content in the flower bud of lily magnolia, belong to field of chemical detection.Aim to provide the method that a kind of short and reproducible ASE HPLC methods of extraction time determine magnelin content in the flower bud of lily magnolia.The method uses ASE methods methanol extraction flower bud of lily magnolia powder, and collects methanol extraction liquid；The content of the magnelin in methanol extraction liquid is determined using HPLC methods.The present invention can replace extraction of the official method to magnelin in the flower bud of lily magnolia, and carry out assay to magnelin in the flower bud of lily magnolia.
Description
Technical field
The invention belongs to the extraction field of magnelin, magnelin contains during especially a kind of ASE-HPLC methods determine the flower bud of lily magnolia
The method of amount.
Background technology
The described method on magnelin in the extraction flower bud of lily magnolia is in version official method in 2015：Take this product meal about
1g, it is accurately weighed, to put in conical flask with cover, precision adds ethyl acetate 20ml, and weighed weight is soaked 30 minutes, ultrasonically treated
(power 250W, frequency 33kHz) 30 minutes, is let cool, then weighed weight, and the weight of less loss is supplied with methyl alcohol, is shaken up, filtration, essence
Close to measure subsequent filtrate 3ml, (100~200 mesh, 2g, internal diameter is 9mm, and wet method dress post uses ethyl acetate to be added in neutral alumina column
5ml prewashing) on, eluted with methyl alcohol 15ml, eluent is collected, in putting 25ml measuring bottles, plus methyl alcohol is to scale, shakes up, filtration, takes continuous
Filtrate, obtains final product.
It is more long the time required to the method and higher to the specialty requirement of technical staff.
The content of the invention
For above-mentioned deficiency, the present invention is intended to provide in a kind of short and reproducible ASE methods extraction flower bud of lily magnolia of extraction time
The method of magnelin, present invention also offers the method for magnelin content in a kind of HPLC methods detection flower bud of lily magnolia.
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 magnelin content, comprises the steps successively in the flower bud of lily magnolia：
Step 1：Using ASE methods methanol extraction flower bud of lily magnolia powder, and collect methanol extraction liquid；
Step 2：The content of the magnelin in methanol extraction liquid is determined using HPLC methods.
Preferably, described step 1 specifically includes following sub-steps：
Step S1：By flower bud of lily magnolia sample comminution, take 1g and be well mixed with 1g diatomite；
Step S2：The mixture of step 1 gained is loaded on and is placed with the ASE abstraction pools of filter membrane and 3g neutral aluminas, plus
Diatomite is extremely parallel with pond mouthful；
Step S3：With methanol extraction, after terminating, with methanol constant volume to 50ml, shake up, filter, take filtrate, you can.
Preferably, the extraction conditions described in step S3 is：Pressure is 1500psi, and temperature is 100 DEG C, and the time is 5min, secondary
Number is 2 times, and flush volume is 60%, and purge time is 60s.
Preferably, the ASE abstraction pools volume described in step S2 is 10ml.
Preferably, the detection parameter of the HPLC methods described in step 2 is：
A) instrument：Agilent1260 Ultra Performance Liquid Chromatography instruments；
B) chromatographic column：Agilent ZORBAX Extend-C18,1.8 μm, 4.6 × 100mm；
C) column temperature：35℃；
D) flow velocity：0.4mL/min；
E) mobile phase：Volume ratio is 35：1：64 acetonitrile-tetrahydrofuran-water；
F) Detection wavelength 278nm；
Number of theoretical plate is calculated by magnelin peak and should be not less than 9000.
Compared with conventional method, tool has the advantage that the present invention：
1st, the present invention improves the viscosity of temperature reduction solvent, reduces solvent into the prevention of sample matrices, increased
Solvent enters the diffusion of sample matrices, reduces the surface tension between solvent and sample matrices, solvent is dissolved the appearance of determinand
Amount increases；
2nd, due to liquid to the solvability of solute much larger than gas to the solvability of solute, therefore the boiling point of extraction liquids
Improved with pressure rise, so that solvent remains at liquid at high temperature under high pressure；
3rd, the present invention has been investigated and has used ethyl acetate, methyl alcohol as Extraction solvent, the as a result extraction of ethyl acetate and methyl alcohol
Effect is basically identical, can not need to be evaporated directly as the solvent of upper HPLC after being extracted due to ethyl acetate, therefore use methyl alcohol more
It is convenient, therefore methyl alcohol is used as Extraction solvent；
4th, the present invention has investigated and has added the neutral alumina of 1g, 2g, 3g, is added without neutral alumina and investigated, as a result
RAD is 1.18%, it was demonstrated that adds neutral alumina little on the influence of its result, but reaches clean-up effect again, so testing
Can be to carry out removal of impurities with 1g neutral aluminas in journey.
Brief description of the drawings
Fig. 1 is to carry out linear regression graph with the concentration of magnelin reference substance (mg)-peak area；
Fig. 2 is the chromatogram of magnelin reference substance；
Fig. 3 is the chromatogram using the magnelin obtained by the extraction of ASE methods；
Fig. 4 is the chromatogram using the magnelin obtained by official method extraction；
Fig. 5 is the investigation analysis chart for adding purification dosage.
Specific embodiment
With reference to specific embodiment, claims of the present invention is described in further detail, but do not constituted
Any limitation of the invention, any limited number of time modification made in the range of the claims in the present invention protection, still in this hair
In bright claims.
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.),
Agilent1260 Ultra Performance Liquid Chromatography instruments.
1.2 reagents：
Water：Meet the one-level water of the regulations of GB/T 6682；
Methyl alcohol (CH4O)：Chromatographically pure (phase chromatography-use).
2nd, method
The preparation of 2.1 reference substance solutions：
Take magnelin reference substance appropriate, it is accurately weighed, plus methyl alcohol is made solution of every 1ml containing 0.1mg, obtains final product.
The preparation of 2.2 need testing solutions：
2.2.1 version official method in 2015：
This product meal about 1g is taken, accurately weighed, in putting conical flask with cover, precision adds ethyl acetate 20ml, weighed weight,
Immersion 30 minutes, ultrasonically treated (power 250W, frequency 33kHz) 30 minutes is let cool, then weighed weight, and less loss is supplied with methyl alcohol
Weight, shake up, filter, precision measures subsequent filtrate 3ml, be added in neutral alumina column (100~200 mesh, 2g, internal diameter is 9mm,
Wet method dress post, with ethyl acetate 5ml prewashing) on, eluted with methyl alcohol 15ml, eluent is collected, in putting 25ml measuring bottles, plus methyl alcohol is extremely
Scale, shakes up, filtration, takes subsequent filtrate, obtains final product.
2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method：
The size-reduced machine of sample is crushed, and takes meal about 1g, accurately weighed, is well mixed with 1g diatomite stand-by, is moved into pre-
First put cellulose filter membrane and 3g neutral aluminas well (chromatography is used)Appropriate diatomite is added in 10ml abstraction pools,
Gently shaking is allowed to Chi Kou in the same horizontal line, tighten and covered on abstraction pool.After extraction terminates, extract is shifted in 50ml
In volumetric flask, with methanol dilution to scale, shake up, filter, take subsequent filtrate, obtain final product.
2.3ASE extraction conditions：Pressure is 1500psi, and temperature is 100 DEG C, and extraction time is 5min, and number of times is 2 times, punching
It is 60% to wash volume, and purge time is 60s.
2.4 chromatographic conditions and system suitability
A) instrument：Agilent1260 Ultra Performance Liquid Chromatography instruments；
B) chromatographic column：Agilent ZORBAX Extend-C18,1.8 μm, 4.6 × 100mm；
C) column temperature：35℃；
D) flow velocity：0.4mL/min；
E) mobile phase：：Acetonitrile-tetrahydrofuran-water (35：1：64)；
F) Detection wavelength 278nm；
Number of theoretical plate is calculated by magnelin peak and should be not less than 9000.
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 1 μ l of need testing solution, liquid chromatograph is injected, determine, obtain final product.
The requirement of 2.6 standard limited values
This product is calculated by dry product, containing magnelin (C23H28O7) 0.40% must not be less than.
2.7th, (external standard method) is calculated
CR in formula --- reference substance solution concentration, unit is micro- gram per liter (mg/L)；
The peak area of AX --- test sample；
AR --- reference substance peak area.
Note：
Should be dismantled using preceding extraction bottom of pond portion and cleaned out, otherwise easily cause pressure instability；
The filter paper of abstraction pool bottom should otherwise cause seepage in sealing ring；
Elastic moderate during abstraction pool dress sample, 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 dry (get rusty easily) in time.
3rd, result
3.1 linear relationships
Take magnelin reference substance appropriate, accurately weighed, in putting brown measuring bottle, plus methyl alcohol is made every 1ml containing magnelin
The solution of 1.0292mg, then the accurate solution 0.1 μ l, 0.2 μ l, 0.5 μ l, 1 μ l, 1.2ul of drawing carries out HPLC measure respectively,
And determined according to the above method, the results detailed in Table 1.
Linear regression is carried out with reference substance concentration (mg)-peak area, Fig. 1 is referred to, regression equation is tried to achieve：Y=1,
323.5649x-37.8978,R2=0.9999, as a result show：Magnelin is in the range of 0.1029-1.2350mg/ml in good
Good linear relationship.
The linear test result of table 1
3.2 replica tests
Take the sample (lot number of identical lot number：The flower bud of lily magnolia -3) about 1g, it is totally 3 parts, accurately weighed, extracted by ASE extracting methods and supplied
Test sample solution, sample size is 1 μ L, and with above-mentioned chromatographic condition parallel test, the content for measuring stachydrine hydrochloride in sample is shown in Table
2, RSD is 2.22%, as a result shows that ASE extracting methods repeatability is good.
The repeated experiment result of table 2
3.3 precision and stability test
Magnelin reference substance solution (1.0292mg/ml) is taken, continuous sample introduction 6 times records peak area, and peak area RSD is
0.5%, Fig. 2 is referred to, as a result show that instrument precision is good.
In addition, seeing Fig. 3 using the chromatogram of the magnelin obtained by the extraction of ASE methods；Using obtained by official method extraction
The chromatogram of magnelin is shown in Fig. 4.
3.4 samples difference instrument extract result and with official method results contrast (3 batches)
Table 3 uses the extraction result of different ASE instruments and official method
Table 4 uses official method and the extraction Comparative result using ASE1 methods
| Pharmacopeia | ASE1 | Average value % | RAD% | |
| The flower bud of lily magnolia -1 | 4.23 | 4.39 | 4.31 | 1.9 |
| The flower bud of lily magnolia -2 | 4.14 | 4.77 | 4.46 | 7.1 |
| The flower bud of lily magnolia -3 | 4.00 | 4.23 | 4.12 | 2.8 |
Table 5 uses official method and the extraction Comparative result using ASE2 methods
| Pharmacopeia | ASE2 | Average value % | RAD% | |
| The flower bud of lily magnolia -1 | 4.23 | 4.35 | 4.29 | 1.4 |
| The flower bud of lily magnolia -2 | 4.14 | 4.86 | 4.50 | 8.0 |
| The flower bud of lily magnolia -3 | 4.00 | 4.32 | 4.16 | 3.9 |
Table 6 uses ASE1 and the extraction Comparative result using ASE2 methods
| ASE1 | ASE2 | Average value % | RAD% | |
| The flower bud of lily magnolia -1 | 4.39 | 4.35 | 4.37 | 0.5 |
| The flower bud of lily magnolia -2 | 4.77 | 4.86 | 4.81 | 0.9 |
| The flower bud of lily magnolia -3 | 4.23 | 4.32 | 4.27 | 1.0 |
4th, discuss：
The investigation of 4.1 Extraction solvents
Investigated in experiment and used ethyl acetate, methyl alcohol as Extraction solvent, the as a result extraction of ethyl acetate and methyl alcohol has been imitated
It is really basically identical, can not need to be evaporated directly as the solvent of upper HPLC after being extracted due to ethyl acetate, therefore use methyl alcohol more square
Just, therefore using methyl alcohol as Extraction solvent.
4.2 investigations for adding purification dosage
Investigated in experiment and added the neutral alumina of 1g, 2g, 3g, be added without neutral alumina and investigated, referred to figure
5, as a result RAD is 1.18%, it was demonstrated that adds neutral alumina little on the influence of its result, but reaches clean-up effect again, so
Can be to carry out removal of impurities with 1g neutral aluminas in process of the test.
It is above-described be only presently preferred embodiments of the present invention, it is all made in the range of the spirit and principles in the present invention appoint
What modification, equivalent and improvement etc., should be included within the scope of the present invention.
Claims (5)
1. a kind of method that ASE-HPLC methods determine magnelin content in the flower bud of lily magnolia, it is characterised in that comprise the steps successively：
Step 1：Using ASE methods methanol extraction flower bud of lily magnolia powder, and collect methanol extraction liquid；
Step 2：The content of the magnelin in methanol extraction liquid is determined using HPLC methods.
2. the method that a kind of ASE-HPLC methods according to claim 2 determine magnelin content in the flower bud of lily magnolia, its feature exists
In described step 1 specifically includes following sub-steps：
Step S1：By flower bud of lily magnolia sample comminution, take 1g and be well mixed with 1g diatomite；
Step S2：The mixture of step 1 gained is loaded on and is placed with the ASE abstraction pools of filter membrane and 3g neutral aluminas, plus diatom
Soil is extremely parallel with pond mouthful；
Step S3：With methanol extraction, after terminating, with methanol constant volume to 50ml, shake up, filter, take filtrate, you can.
3. the method that a kind of ASE-HPLC methods according to claim 2 determine magnelin content in the flower bud of lily magnolia, its feature exists
In the extraction conditions described in step S3 is：Pressure is 1500psi, and temperature is 100 DEG C, and the time is 5min, and number of times is 2 times, is rinsed
Volume is 60%, and purge time is 60s.
4. the method that a kind of ASE-HPLC methods according to claim 2 determine magnelin content in the flower bud of lily magnolia, its feature exists
In the ASE abstraction pools volume described in step S2 is 10ml.
5. the method that a kind of ASE-HPLC methods according to claim 1 determine magnelin content in the flower bud of lily magnolia, its feature exists
In the detection parameter of the HPLC methods described in step 2 is：
A) instrument：Agilent1260 Ultra Performance Liquid Chromatography instruments；
B) chromatographic column：Agilent ZORBAX Extend-C18,1.8 μm, 4.6 × 100mm；
C) column temperature：35℃；
D) flow velocity：0.4mL/min；
E) mobile phase：Volume ratio is 35：1：64 acetonitrile-tetrahydrofuran-water；
F) Detection wavelength 278nm；
Number of theoretical plate is calculated by magnelin peak and should be not less than 9000.
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