CN106918666A - A kind of method that ASE HPLC methods determine the curculigoside content in thizoma curculiginis - Google Patents
A kind of method that ASE HPLC methods determine the curculigoside content in thizoma curculiginis Download PDFInfo
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- CN106918666A CN106918666A CN201710232491.6A CN201710232491A CN106918666A CN 106918666 A CN106918666 A CN 106918666A CN 201710232491 A CN201710232491 A CN 201710232491A CN 106918666 A CN106918666 A CN 106918666A
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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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- 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
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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 Curculigoside in Crude Medicine Curculigo orchioides content, belong to field of chemical detection.A kind of method that extraction effect is good and accuracy of detection is high is aimed to provide, the method uses ASE methods methanol extraction Common Curculigo Rhizome, and collects methanol extraction liquid；Curculigoside content in methanol extraction liquid is determined using HPLC methods, wherein, the mobile phase for using is the phosphoric acid of acetonitrile 0.1%.The alternative official method of the present invention carries out assay and quality control to the curculigoside in thizoma curculiginis.
Description
Technical field
The side of Curculigoside in Crude Medicine Curculigo orchioides content is determined the invention belongs to field of chemical detection, especially a kind of ASE-HPLC methods
Method.
Background technology
Pharmacopeia in 2015 have recorded and take this product powder (crossing No. three sieves) about 1g, and accurately weighed, precision adds methyl alcohol 50ml, claims
Determine weight, be heated to reflux 2 hours, take out, let cool, then weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter.It is accurate
Subsequent filtrate 20ml is measured, is evaporated, residue adds methyl alcohol to dissolve, be transferred in 10ml measuring bottles, plus methyl alcohol is to scale, shakes up, filtration, takes
Subsequent filtrate, obtains final product.
Liquid chromatographic detection condition is：With octadecylsilane chemically bonded silica as filler；It is molten with the phosphoric acid of acetonitrile -0.1%
Liquid (21：79) it is mobile phase；Detection wavelength is 285nm.Number of theoretical plate is calculated by curculigoside peak and should be not less than 3000.
The method operating process of pharmacopeia is complicated, and the operative skill requirement to operating personnel is high.
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 Curculigoside in Crude Medicine Curculigo orchioides content,
ASE is extracted and is combined with HPLC detections by the method, and the volume ratio of mobile phase in official method is optimized, to reach
To more preferable experiment effect.
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 Curculigoside in Crude Medicine Curculigo orchioides content, comprises the steps successively：
Step 1：Using ASE methods methanol extraction Common Curculigo Rhizome, and collect methanol extraction liquid；
Step 2：Using HPLC methods determine methanol extraction liquid in curculigoside content, wherein, the mobile phase for using for acetonitrile-
0.1% phosphoric acid.
Preferably, described step 1 specifically includes following sub-steps：
Step S1：By thizoma curculiginis sample comminution, No. three sieves are crossed, take 1g and mix with 0.5g diatomite；
Step S2：By the mixture of step 1 gained loaded on being placed with the 10ml ASE abstraction pools of filter membrane, plus diatomite to
Pond mouthful is parallel；
Step S3：With methanol extraction, after extraction terminates, extract is settled to 50ml with methyl alcohol, under 15000r/min
Centrifugation 5min, takes supernatant.
Preferably, the extraction parameters described in step S3 are：Temperature is 90 DEG C, and the time is 5min, and number of times is 2 times, rinses body
Product is 100%, and purge time is 60s.
Preferably, the detection parameter of the HPLC methods described in step 2 is：Chromatographic column is Thermo Syncronis C18,3*
100mm, 3 μm；Column temperature is 40 DEG C；Flow velocity is 0.5mL/min；Mobile phase is that volume ratio is equal to 19：81 phosphoric acid of acetonitrile -0.1%；
Detection wavelength is 285nm.
The present invention compared with conventional method, with advantages below：
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, determine curculigoside reference substance using detection method of the present invention is in 0.0102~0.0612 μ g ranges
Good linear relationship；
4th, ASE extracting methods repeatability of the present invention is good；
5th, the present invention has investigated methyl alcohol, ethyl acetate, ethanol, how much is preferable according to selecting with recovery rate height, impurity
Solvent, as a result shows methyl alcohol recovery rate highest, successively to ethanol, ethyl acetate；Ethanol recovery rate is higher than pharmacopeia, but peak type is poor；
Acetic acid ethyl acetate extract could be determined after need to being evaporated constant volume, be operated relatively complicated；So selection methyl alcohol is used as Extraction solvent；
6th, the present invention uses the horizontal quadrature experiment investigation of 3 factor 3 three factors larger on extraction efficiency influence：Extraction
Temperature, static extracting time, cycle-index, by result of the test, it is determined that preferably ASE parameters, as a result show：Extraction temperature pair
The content influence of curculigoside is maximum, next to that cycle-index and static extracting time；Finally determine that preferable ASE parameters are extraction temperature
90 DEG C of degree, static extracting time 5min, circulation is extracted 2 times.
Brief description of the drawings
Fig. 1 is to carry out linear regression graph with concentration (μ the g)-peak area of curculigoside reference substance；
Fig. 2 is the chromatogram of curculigoside reference substance；
Fig. 3 is the sample chromatogram figure determined using official method；
Fig. 4 is the sample chromatogram figure determined using ASE-HPLC methods.
Specific embodiment
With reference to specific embodiment, claim of the invention is described in further detail, but do not constitute it is right
Any limitation of the invention, any limited number of time modification made in the claims in the present invention protection domain, still of the invention
In 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.), Thermo
U3000UHPLC liquid chromatographs.
1.2 reagents：
Water：Meet the one-level water of the regulations of GB/T 6682；
Acetonitrile (C2H3N)：Chromatographically pure；
Phosphoric acid (H3PO4)：Analysis is pure
2nd, method
The preparation of 2.1 reference substance solutions：
Take curculigoside reference substance appropriate, it is accurately weighed, plus methyl alcohol is made solution of every 1ml containing 70 μ g, obtains final product.
The preparation of 2.2 need testing solutions：
2.2.1《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：
This product powder (crossing No. three sieves) about 1g is taken, accurately weighed, precision adds methyl alcohol 50ml, and weighed weight is heated to reflux 2
Hour, take out, let cool, then weighed weight, the weight of less loss is supplied with methyl alcohol, shake up, filter.Precision measures subsequent filtrate 20ml,
It is evaporated, residue adds methyl alcohol to dissolve, is transferred in 10ml measuring bottles, plus methyl alcohol is to 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 crosses No. three sieves, and about 1g is accurately weighed, is well mixed with 0.5g diatomite, stand-by, moves
Enter to putting filter membrane well in advanceAppropriate diatomite is added in 10ml abstraction pools, gently shaking is allowed to Chi Kou same
On one horizontal line, tighten and covered on abstraction pool.After extraction terminates, extract is shifted in 50ml volumetric flasks, with methanol dilution extremely
Scale, 5min is centrifuged under 15000r/min, takes supernatant, is determined into LC.
2.3ASE extraction conditions：Temperature is 90 DEG C, and the time is 5min, and number of times is 2 times, and flush volume is 100%, during purging
Between be 60s.
2.4 chromatographic conditions and system suitability
Chromatographic column is Thermo Syncronis C18,3*100mm, 3 μm；Column temperature is 40 DEG C；Flow velocity is 0.5mL/min；
Mobile phase is that volume ratio is equal to 19：81 phosphoric acid of acetonitrile -0.1%；Detection wavelength is 285nm；With octadecylsilane bonded silica
Glue is filler；Number of theoretical plate is calculated by curculigoside peak and should be not less than 3000.
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, determine, obtain final product.
The requirement of 2.6 standard limited values
This product is calculated by dry product, containing curculigoside (C22H26O11) 0.10% 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)；
AX--- the peak area of 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 curculigoside reference substance solution (concentration：0.0204mg/ml), it is accurate respectively to draw the solution 0.5 μ l, 1 μ l, 1.5 μ
L, 2 μ l, 3 μ l are determined into LC, and are determined according to the above method, the results detailed in Table 1.
Linear regression is carried out with concentration (μ g)-peak area, Fig. 1 is referred to, regression equation is tried to achieve：Y=1898.1x+2.2775,
R2=0.9999.
Result shows：Curculigoside is in good linear relationship in 0.0102~0.0612 μ g ranges.
The linear test result of table 1
3.2 replica tests
Take the sample (lot number of identical lot number：20150701) 1g, it is totally 6 parts, accurately weighed, extracted by ASE extracting methods and supplied
Test sample solution, sample size is 2 μ l, and with above-mentioned chromatographic condition parallel test, the content for measuring curculigoside in sample is shown in Table 2, RSD
It is 1.6%, experiment shows that ASE extracting methods repeatability is good.
The replica test result of table 2
3.3 precision
Curculigoside reference substance solution (0.0204mg/ml) is taken, continuous sample introduction 6 times records peak area, and peak area RSD is
0.5%, show that instrument precision is good.
3.4 samples difference instrument extract result and with official method results contrast (4 batches)
The different instruments of table 3 extract results contrast
The ASE of table 4 extracts results contrast with pharmacopeia
4th, discuss
The selection of 4.1 ASE Extraction solvents
Curculigoside is the principle active component of thizoma curculiginis, dissolves in warm water, methyl alcohol, ethanol, n-butanol, ethyl acetate etc..This
Methyl alcohol, ethyl acetate, ethanol have been investigated in experiment, how much are that foundation selects preferred solvents with recovery rate height, impurity.Result shows
Methyl alcohol recovery rate highest, successively to ethanol, ethyl acetate.Ethanol recovery rate is higher than pharmacopeia, but peak type is poor；Acetic acid ethyl acetate extract
Could be determined after constant volume need to be evaporated, operated relatively complicated.
So selection methyl alcohol the results are shown in Table 5 as Extraction solvent：
The Extraction solvent of table 5 investigates result
| Extraction solvent | Determine content (%) |
| Methyl alcohol | 0.0307 |
| Ethanol | 0.0288 |
| Ethyl acetate | 0.0272 |
The optimization of 4.2 ASE extraction conditions
This experiment uses the horizontal quadrature experiment investigation of 3 factor 3 three factors larger on extraction efficiency influence：Extraction temperature
Degree, static extracting time, cycle-index, by result of the test, it is determined that preferably ASE parameters, refer to table 6, table 7.
The factor level of table 6
The orthogonal experiment range analysis result of table 7
By range analysis as can be seen that extraction temperature influences maximum to the content of curculigoside, next to that cycle-index and quiet
State extraction time；Preferable ASE parameters are finally determined for 90 DEG C of extraction temperature, and static extracting time 5min, circulation is extracted 2 times.
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 (4)
1. a kind of method that ASE-HPLC methods determine Curculigoside in Crude Medicine Curculigo orchioides content, it is characterised in that comprise the steps successively：
Step 1：Using ASE methods methanol extraction Common Curculigo Rhizome, and collect methanol extraction liquid；
Step 2：Using HPLC methods determine methanol extraction liquid in curculigoside content, wherein, the mobile phase for using for acetonitrile-
0.1% phosphoric acid.
2. the method that a kind of ASE-HPLC methods according to claim 1 determine Curculigoside in Crude Medicine Curculigo orchioides content, it is characterised in that
Described step 1 specifically includes following sub-steps：
Step S1：By thizoma curculiginis sample comminution, No. three sieves are crossed, take 1g and mix with 0.5g diatomite；
Step S2：By the mixture of step 1 gained loaded on being placed with the 10ml ASE abstraction pools of filter membrane, plus diatomite to and Chi Kou
It is parallel；
Step S3：With methanol extraction, after extraction terminates, extract is settled to 50ml with methyl alcohol, is centrifuged under 15000r/min
5min, takes supernatant.
3. the method that a kind of ASE-HPLC methods according to claim 2 determine Curculigoside in Crude Medicine Curculigo orchioides content, it is characterised in that
Extraction parameters described in step S3 are：Temperature is 90 DEG C, and the time is 5min, and number of times is 2 times, and flush volume is 100%, during purging
Between be 60s.
4. the method that a kind of ASE-HPLC methods according to claim 1 determine Curculigoside in Crude Medicine Curculigo orchioides content, it is characterised in that
The detection parameter of the HPLC methods described in step 2 is：Chromatographic column is Thermo Syncronis C18,3*100mm, 3 μm；Column temperature is
40℃；Flow velocity is 0.5mL/min；Mobile phase is that volume ratio is equal to 19：81 phosphoric acid of acetonitrile -0.1%；Detection wavelength is 285nm.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105929090A (en) * | 2016-04-22 | 2016-09-07 | 广西壮族自治区梧州食品药品检验所 | Method for extracting curculigoside in Curculigo orchioides |
| CN105954387A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Determination method for curculigoside in Curculigo orchioides |
| CN105954407A (en) * | 2016-04-27 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for determination of curculigine in curculigo orchioides |
| CN105954440A (en) * | 2016-04-27 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for extracting curculigine in curculigo orchioides |
-
2017
- 2017-04-11 CN CN201710232491.6A patent/CN106918666A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105929090A (en) * | 2016-04-22 | 2016-09-07 | 广西壮族自治区梧州食品药品检验所 | Method for extracting curculigoside in Curculigo orchioides |
| CN105954387A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Determination method for curculigoside in Curculigo orchioides |
| CN105954407A (en) * | 2016-04-27 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for determination of curculigine in curculigo orchioides |
| CN105954440A (en) * | 2016-04-27 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for extracting curculigine in curculigo orchioides |
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Application publication date: 20170704 |