CN107014927A - A kind of method that ASE HPLC methods determine Determination of hesperidin in pericarpium citri reticulatae - Google Patents
A kind of method that ASE HPLC methods determine Determination of hesperidin in pericarpium citri reticulatae Download PDFInfo
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- CN107014927A CN107014927A CN201710263192.9A CN201710263192A CN107014927A CN 107014927 A CN107014927 A CN 107014927A CN 201710263192 A CN201710263192 A CN 201710263192A CN 107014927 A CN107014927 A CN 107014927A
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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
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- 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 Determination of hesperidin in pericarpium citri reticulatae, this method extraction time is short and accuracy of detection is high.Belong to field of chemical detection.The present invention collects methanol extraction liquid using ASE methods extraction Dried Tangerine Peel；The content of aurantiamarin in methanol extraction liquid is determined using HPLC methods.The present invention can be extracted and assay instead of official method to the aurantiamarin in dried orange peel.
Description
Technical field
The invention belongs to field of chemical detection, especially a kind of ASE-HPLC methods determine the side of Determination of hesperidin in pericarpium citri reticulatae
Method.
Background technology
《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：This product coarse powder about 1g is taken, it is accurately weighed, put Soxhlet and carry
Take in device, plus petroleum ether (60~90 DEG C) 80ml, it is heated to reflux 2~3 hours, discards petroleum ether, the dregs of a decoction is volatilized, plus methanol
80ml, reheating is back to extracting liquid colourless, lets cool, and filters, filtrate is put in 100ml measuring bottles, with a small amount of methanol fraction
Secondary washing container, washing lotion is filtered in same measuring bottle, plus methanol is to scale, shakes up, and produces.
This method return time is longer, 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 Determination of hesperidin in pericarpium citri reticulatae,
This method extraction time is short 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 Determination of hesperidin in pericarpium citri reticulatae, comprises the steps successively：
Step 1：Dried Tangerine Peel is extracted using ASE methods, and collects methanol extraction liquid；
Step 2：The content of aurantiamarin in methanol extraction liquid is determined using HPLC methods.
Preferably, the extraction described in step 1 includes first using petroleum ether extraction, again with methanol extraction.
Preferably, described step 1 includes following sub-steps：
Step S1：By dried orange peel sample comminution, 1g is taken to be mixed with 1g diatomite；
Step S2：By the mixture obtained by step 1 loaded on being placed with the ASE abstraction pools of fibrous filter membrane, plus diatomite to
Pond mouthful is parallel；
Step S3：Petroleum ether extraction is first used, extract is discarded；Again with methanol is extracted as extract, after extraction terminates, and is used
Methanol is settled to 100ml, you can.
Preferably, the temperature in use of the petroleum ether described in step S3 is 60~90 DEG C.
Preferably, the parameter of the use petroleum ether extraction described in step S3 is：Temperature is 75 DEG C, and the time is 3min, number of times
For 1 time, flush volume is 60%, and purge time is 60s；
Preferably, the parameter of the use methanol extraction described in step S3 is：Temperature is 100 DEG C, and the time is 8min, and number of times is
3 times, flush volume is 60%, and purge time is 60s.
Preferably, the detection parameter of the HPLC methods described in step 1 is：Chromatographic column is ACE, UltraCore Super C18；
Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is methanol-acetic acid-water；Detection wavelength is 283nm.
Preferably, the specification of described chromatographic column is 2.5 μm, 2.1*75mm.
Preferably, described mobile phase is that volume ratio is equal to 35:4:61 methanol-acetic acid-water.
The present invention is compared with conventional method, with advantages below：
The present invention refers to official method, first with petroleum ether (60~90 DEG C) removal of impurities, and again with methanol is extracted, and obtains good reality
Test result.
The present invention uses 3 factor 3 horizontal quadrature experimental program L9 (33) experiment arrangement, has investigated using methanol as extraction
Extraction temperature, static extracting time, cycle-index during solvent, influence of 3 factors to aurantiamarin in accelerated solvent extraction dried orange peel,
It is determined that extracting the optimal Accelerate solvent extraction technique of aurantiamarin from dried orange peel.
Brief description of the drawings
Fig. 1 is the linear regression graph carried out with the absolute magnitude (mg) of aurantiamarin reference substance sample introduction-peak area；
Fig. 2 is the chromatogram of aurantiamarin reference substance；
Fig. 3 is the chromatogram using the need testing solution obtained by ASE methods；
Fig. 4 is the chromatogram using the need testing solution obtained by official method.
Embodiment
With reference to embodiment, the claim to the present invention is described in further detail, but is not constituted pair
Any limitation of the present invention, any limited number of time modification made in the claims in the present invention protection domain, still the present invention's
In 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.), Agilent
1290 liquid chromatographs
1.2 reagent：Water：Meet one-level water as defined in GB/T 6682；
Methanol (CH4O)：Chromatographically pure (phase chromatography-use).
2nd, method
The preparation of 2.1 reference substance solutions：Take aurantiamarin reference substance appropriate, it is accurately weighed, plus methanol is made every 1ml and contains
0.4mg solution, is produced.
The preparation of 2.2 need testing solutions
2.2.1《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：
This product coarse powder about 1g is taken, it is accurately weighed, put in apparatus,Soxhlet's, plus petroleum ether (60~90 DEG C) 80ml, heat back
Stream 2~3 hours, discards petroleum ether, the dregs of a decoction are volatilized, plus methanol 80ml, and reheating is back to extracting liquid colourless, lets cool, and filters, filter
Liquid is put in 100ml measuring bottles, with a small amount of methanol fraction
Secondary washing container, washing lotion is filtered in same measuring bottle, plus methanol is to scale, shakes up, and produces.
2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method：
The size-reduced machine of sample is crushed, and takes coarse powder about 1g, accurately weighed, is well mixed with about 1g diatomite, stand-by, is moved into
Appropriate diatomite is added in the ASE 10ml abstraction pools for putting fibrous filter membrane well in advance, gently shaking is allowed to Chi Kou in same water
On horizontal line, tighten and covered on abstraction pool, be put into rapid extracting device and extracted.First with (60~90 DEG C) extractions of petroleum ether, discard
Extract；Again with methanol is extracted as extract, and after extraction terminates, extract is transferred in 100ml measuring bottles, plus methanol
Scale is diluted to, is shaken up, is filtered, is taken subsequent filtrate, produce.
2.3 ASE extraction conditions
The parameter of described first time extraction step is：Temperature is 75 DEG C, and the time is 3min, and number of times is 1 time, flush volume
For 60%, purge time is 60s；
The parameter of second described of extraction step is：Temperature is 100 DEG C, and the time is 8min, and number of times is 3 times, rinses body
Product is 60%, and purge time is 60s.
2.4 chromatographic conditions and system suitability
A) instrument：Agilent 1290；
B) chromatographic column：ACE, UltraCore Super C18,2.5 μm, 2.1*75mm；
C) column temperature：40℃；
D) flow velocity：0.3mL/min；
E) mobile phase：：Methanol-acetic acid-water (35:4:61)；
F) Detection wavelength：283nm；
Using octadecylsilane chemically bonded silica as filler；Number of theoretical plate is calculated by aurantiamarin peak should be not less than 2000.
2.5 determination method
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, determines, produces.
The requirement of 2.6 standard limited values
This product is calculated by dry product, containing aurantiamarin (C28H34O15) 3.5% 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：
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 relationship
Aurantiamarin reference substance is taken, it is accurately weighed, plus solution of every 1ml containing about 0.4mg is made in methanol, produces, then distinguishes
Precision is drawn the solution 0.2 μ l, 0.5 μ l, 0.8 μ l, 1.0 μ l, 1.5 μ l, 2.0 μ l and determined into LC, and is surveyed according to the above method
It is fixed, the results detailed in Table 1.
Linear regression is carried out with sample introduction absolute magnitude (mg)-peak area, regression equation is tried to achieve：Aurantiamarin y=4794.1314x+
41.4331, R2=1.0000, aurantiamarin is in good linear relationship in 0.0822~0.8215mg encloses, and refers to Fig. 1.
The Linear Experiment result of table 1
| Sample introduction absolute magnitude (mg) | First pin peak area | Second pin peak area | Average peak area |
| 0.0822 | 421.79071 | 437.20343 | 429.49707 |
| 0.2054 | 1032.55029 | 1021.52869 | 1027.03949 |
| 0.3286 | 1622.84851 | 1629.01575 | 1625.93213 |
| 0.4108 | 2010.51843 | 2001.51160 | 2006.01502 |
| 0.6161 | 3003.70654 | 2998.18799 | 3000.94727 |
| 0.8215 | 3977.98218 | 3971.10645 | 3974.54432 |
3.2 precision and stability experiment
Aurantiamarin reference substance solution (0.3286mg/ml) is taken, continuous sample introduction 6 times records peak area, and peak area RSD is
0.7%, show that instrument precision is good.
Same need testing solution is taken, is placed at room temperature, is determined in accordance with the law respectively at 0,2,4,6,8,12h.As a result aurantiamarin peak
The RSD of area is 0.3%, shows that need testing solution is stable in 12h.
Measured chromatogram refers to Fig. 2 to Fig. 4, and wherein Fig. 2 is the chromatogram of aurantiamarin reference substance, and Fig. 3 is to use ASE
The chromatogram of need testing solution obtained by method, Fig. 4 is the chromatogram using the need testing solution obtained by official method.
3.3 repeated experiment
The sample 1g of identical lot number (lot number 20150801) is taken, it is totally 9 parts, accurately weighed, extracted by above-mentioned ASE extracting methods
Need testing solution, sample size is 1 μ l, and with above-mentioned chromatographic condition parallel test, the content for measuring aurantiamarin in sample is shown in Table 2,
RSD is 1.9%, as a result shows that ASE extracting methods repeatability is good.
The repeated experiment result of table 2
3.4 samples difference instrument extract result and with official method results contrast (3 batches)
The extraction results contrast of the difference ASE instrument of table 3
The extraction results contrast of the ASE methods of table 4 and official method
4th, discuss：
The selection of 4.1 ASE Extraction solvents：
The present invention refers to official method, first with petroleum ether (60~90 DEG C) removal of impurities, and again with methanol is extracted, and obtains good reality
Test result.
The optimization of 4.2 ASE extraction conditions
Using 3 factor 3 horizontal quadrature experimental program L9 (33) experiment arrangement, when having investigated using methanol as extractant
Extraction temperature, static extracting time, cycle-index, influence of 3 factors to aurantiamarin in accelerated solvent extraction dried orange peel (are shown in Table
5).It is determined that extracting the optimal Accelerate solvent extraction technique of aurantiamarin from dried orange peel, 6 are the results are shown in Table.
The factor level of table 5
The orthogonal experiment range analysis result of table 6
Extracting temperature influences maximum to the content of aurantiamarin it can be seen from range analysis, next to that the static extracting time
And extraction time.It is final to determine that optimum extraction process is combined as 100 DEG C of Extracting temperature, extraction time 8min, 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 modifications, equivalent substitutions and improvements etc., should be included in the scope of the present invention.
Claims (9)
1. a kind of method that ASE-HPLC methods determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that comprise the steps successively：
Step 1：Dried Tangerine Peel is extracted using ASE methods, and collects methanol extraction liquid；
Step 2：The content of aurantiamarin in methanol extraction liquid is determined using HPLC methods.
2. the method that a kind of ASE-HPLC methods according to claim 1 determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that
Extraction described in step 1 includes first using petroleum ether extraction, again with methanol extraction.
3. the method that a kind of ASE-HPLC methods according to claim 1 determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that
Described step 1 includes following sub-steps：
Step S1：By dried orange peel sample comminution, 1g is taken to be mixed with 1g diatomite；
Step S2：By the mixture obtained by step 1 loaded on being placed with the ASE abstraction pools of fibrous filter membrane, plus diatomite to and Chi Kou
It is parallel；
Step S3：Petroleum ether extraction is first used, extract is discarded；Again with methanol is extracted as extract, after extraction terminates, uses methanol
It is settled to 100ml, you can.
4. the method that a kind of ASE-HPLC methods according to claim 3 determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that
The temperature in use of petroleum ether described in step S3 is 60~90 DEG C.
5. the method that a kind of ASE-HPLC methods according to claim 3 determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that
The parameter of use petroleum ether extraction described in step S3 is：Temperature is 75 DEG C, and the time is 3min, and number of times is 1 time, and flush volume is
60%, purge time is 60s.
6. the method that a kind of ASE-HPLC methods according to claim 3 determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that
The parameter of use methanol extraction described in step S3 is：Temperature is 100 DEG C, and the time is 8min, and number of times is 3 times, and flush volume is
60%, purge time is 60s.
7. the method that a kind of ASE-HPLC methods according to claim 1 determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that
The detection parameter of HPLC methods described in step 1 is：Chromatographic column is ACE, UltraCore Super C18；Column temperature is 40 DEG C；Flow velocity
For 0.3mL/min；Mobile phase is methanol-acetic acid-water；Detection wavelength is 283nm.
8. the method that a kind of ASE-HPLC methods according to claim 7 determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that
The specification of described chromatographic column is 2.5 μm, 2.1*75mm.
9. the method that a kind of ASE-HPLC methods according to claim 7 determine Determination of hesperidin in pericarpium citri reticulatae, it is characterised in that
Described mobile phase is that volume ratio is equal to 35:4:61 methanol-acetic acid-water.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101704867A (en) * | 2009-11-03 | 2010-05-12 | 国家海洋局第三海洋研究所 | Method for preparing naringin or hesperidin |
| CN102670951A (en) * | 2011-03-16 | 2012-09-19 | 河北以岭医药研究院有限公司 | Method for determining content of hesperidin in Chinese medicinal composition |
| CN104892701A (en) * | 2015-05-12 | 2015-09-09 | 广西壮族自治区梧州食品药品检验所 | Method used for extracting hesperidin from pills used for promoting digestion |
| CN105954405A (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 pectolinarin in buddleja officinalis |
-
2017
- 2017-04-17 CN CN201710263192.9A patent/CN107014927A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101704867A (en) * | 2009-11-03 | 2010-05-12 | 国家海洋局第三海洋研究所 | Method for preparing naringin or hesperidin |
| CN102670951A (en) * | 2011-03-16 | 2012-09-19 | 河北以岭医药研究院有限公司 | Method for determining content of hesperidin in Chinese medicinal composition |
| CN104892701A (en) * | 2015-05-12 | 2015-09-09 | 广西壮族自治区梧州食品药品检验所 | Method used for extracting hesperidin from pills used for promoting digestion |
| CN105954405A (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 pectolinarin in buddleja officinalis |
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