CN103235070A - High performance liquid chromatography method for determining content of resveratrol in polygonum cuspidatum - Google Patents
High performance liquid chromatography method for determining content of resveratrol in polygonum cuspidatum Download PDFInfo
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- CN103235070A CN103235070A CN2013101759749A CN201310175974A CN103235070A CN 103235070 A CN103235070 A CN 103235070A CN 2013101759749 A CN2013101759749 A CN 2013101759749A CN 201310175974 A CN201310175974 A CN 201310175974A CN 103235070 A CN103235070 A CN 103235070A
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Abstract
The invention discloses a high performance liquid chromatography method for determining the content of resveratrol in polygonum cuspidatum. A chromatographic column is Thermo BDS-C18 (250mm*4.6mm, 5 mu m); a mobile phase is of methanol and water with the volume ratio of 55: 45; the detection temperature is 30 DEG C; the sample injection volume is 5 mu l; the flow rate is 0.8ml/min; a detector is an ultraviolet detector; and the detection wavelength is 306nm. The resveratrol has a good linear relation with a peak area in a concentration range of 10 mu g/ml-200 mu g/ml, and the linear correlation coefficient r is 1. The high performance liquid chromatography method for detecting the content of the resveratrol has the technical characteristics of good separation effect, good precision, high sensitivity, simplicity, convenience and rapidness in analysis and the like.
Description
Technical field
The present invention relates to the high-performance liquid chromatogram determination method of Resveratrol content in a kind of giant knotweed.
Background technology
Resveratrol (Resveratrol) is the natural diphenylethylene polyphenol substance (structural formula is seen accompanying drawing 1) that produces in the plant, and molecular formula is C
14H
12O
3, chemistry is by name 3,4 ', 5-resveratrol, resveratrol are a kind of biological very strong natural polyphenol class materials, are called resvertrol again.Resveratrol is present in the plants such as giant knotweed, grape, and the content of resveratrol is higher than other plants such as grape far away in the giant knotweed.Along with to the going deep into of resveratrol research, its pharmacological action widely is found gradually, and as anti-inflammatory, antitumor, antibiotic, antiviral, anti-ageing and immunological regulation etc., he can also alleviate the injuries of tissues and organs that multiple factor causes and protect hepatocellular effect.That the effect of resveratrol aspect cardiovascular system comprises is anti-oxidant, remove the free radical effect, suppress platelet aggregation, anti-apoptotic, adjusting blood fat, prevent multiple pharmacological effect such as atherosclerotic and pre-anti-cancer.The result of study of United States Department of Agriculture shows, resveratrol has tangible prevention effect to hormone-dependent neoplasm, also can prevention effect be arranged to osteoporosis, acne and senile dementia, the anti-ageing enzyme of a kind of monomer of inside of human body is worked, and then performance prevents various age-related disease, prolongs the latent effect of expected life.
Since the nineties in 20th century, health-care effect, the detection method of resveratrol in wines have been carried out big quantity research abroad, the domestic report that Resveratrol content in the giant knotweed is measured of not seeing as yet, and in sample concentration of the present invention and peak area linear relationship, I to the 10 μ g/ml of the concentration range of resveratrol, maximum can reach 1000 μ g/ml, and concentration range is bigger in the linear relationship; The present invention also has good separating effect, measures accurately, and is highly sensitive, analyzes the easy quick technical characterstic that waits, and as seen, the method for setting up Resveratrol content in the stable HPLC detection giant knotweed at present is very crucial, has very important meaning.
Summary of the invention
The purpose of this invention is to provide a kind of easy, good separating effect, highly sensitive, precision and the higher Resveratrol content detection method of accuracy.For reaching above-mentioned purpose, the present invention has adopted following technical scheme:
The percent by volume of methyl alcohol was decremented to 50% by 80% during the present invention selected for use and flows mutually, and the particular methanol volume fraction is 55%; The percent by volume of ultrapure water is incremented to 50% by 20%, and the volume fraction of preferred ultrapure water is 45%.
In the HPLC method of the present invention, its characteristics are to select for use above-mentioned mobile phase, by above-mentioned mobile phase gained resveratrol sample collection of illustrative plates peak shape (collection of illustrative plates is seen accompanying drawing 2) and separating effect are preferably arranged.
HPLC analytical approach sample size of the present invention is 5 μ l, utilize standard solution repeatedly sample introduction carry out sample introduction precision test, the sample introduction number of times is 6 times, the RSD value is 0.02%, sample introduction precision is very good.
Description of drawings
Fig. 1 is the resveratrol structural formula
Fig. 2 detects collection of illustrative plates for resveratrol HPLC of the present invention
Fig. 3 is resveratrol standard specimen concentration and peak area linear relationship
Fig. 4 is the variation of the indoor placement different time of resveratrol absorbance
Fig. 5 is that resveratrol absorbance under different temperatures changes
Embodiment
Further specify labor method of the present invention in the following embodiments, but the present invention is not limited to following labor method.
The efficient liquid-phase chromatography method-1 that Resveratrol content is measured
Chromatographic condition: adopt U.S. Agilent-1260 high performance liquid chromatograph, Chemstation Edition chromatographic work station
Chromatographic column: Thermo BDS-C18 (250mm * 4.6mm, 5 μ m)
Phase flows: methyl alcohol: water=55: 45 (volume ratio)
Detected temperatures: 30 ℃
Detecting device: UV-detector
Detect wavelength: 306nm
Vacuum pump: SHB-Ш circulation ability of swimming vacuum pump
Electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd. provides, and specification is 100,000/
The preparation of the phase that flows: flow according to methyl alcohol: water=55: 45 (volume ratio) mixes, and 0.2 μ m aperture filtrator filters, the degassing of SCQ-250W ultrasonic echography.
Adopt external standard method, need respectively standard items and testing sample solution to be measured.
The standard solution preparation: precision takes by weighing 50mg resveratrol standard items, place the brown volumetric flask of 50ml, add and be settled to scale mark with mobile phase dilution again after a small amount of mutually ultrasonic 5seconds of flowing dissolves sample fully, as storing solution, precision is measured storing solution 1ml again, place the brown volumetric flask of 10ml, adding flows is settled to scale mutually.
The testing sample preparation: precision takes by weighing the testing sample with the standard items equivalent weight, places the brown volumetric flask of 50ml, adds to be settled to scale mark with mobile phase dilution again after a small amount of mutually ultrasonic 5seconds of flowing dissolves sample fully.Precision is measured 1ml again, and solution places the brown volumetric flask of 10ml, and adding flows is settled to scale mutually.
According to the cubage formula:
Bring data into and can obtain Resveratrol content.
Above standard items and testing sample are all prepared 3 parts, and absolute error is no more than 0.2%.
The efficient liquid-phase chromatography method-2 that Resveratrol content is measured
Chromatographic condition: adopt U.S. LabAlliance high performance liquid chromatograph, EZChrom Elite Client chromatographic work station
Chromatographic column: SMT-SAM-C18 (250mm * 4.6mm, 5 μ m)
Phase flows: methyl alcohol: water=55: 45 (volume ratio)
Detected temperatures: 30 ℃
Detecting device: SPECTRA STSTEM, UV6000LP UV-detector
Detect wavelength: 306nm
Vacuum pump: SHB-Ш circulation ability of swimming vacuum pump
Electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd. provides, and specification is 100,000/
The preparation of the phase that flows: flow according to methyl alcohol: water=60: 40 (volume ratio) mixes, and 0.2 μ m aperture filtrator filters, the degassing of SCQ-250W ultrasonic echography.
Adopt external standard method, need respectively standard items and testing sample solution to be measured.
The standard solution preparation: precision takes by weighing the 50mg standard items, place the brown volumetric flask of 50ml, add and be settled to scale mark with mobile phase dilution again after the ultrasonic 5seconds of small amount of methanol dissolves sample fully, as storing solution, precision is measured storing solution 1ml again, place the brown volumetric flask of 10ml, adding flows is settled to scale mutually.
The testing sample preparation: precision takes by weighing the testing sample with the standard items equivalent weight, places the brown volumetric flask of 50ml, adds to be settled to scale mark with mobile phase dilution again after the ultrasonic 5seconds of small amount of methanol dissolves sample fully.Precision is measured 1ml again, and solution places the brown volumetric flask of 10ml, and adding flows is settled to scale mutually.
According to the cubage formula:
Bring data into and can obtain Resveratrol content.
Above standard items and testing sample are all prepared 3 parts, and absolute error is no more than 0.2%.
Embodiment 3
The efficient liquid-phase chromatography method-3 that Resveratrol content is measured
Chromatographic condition: adopt U.S. Agilent-1260 high performance liquid chromatograph, Chemstation Edition chromatographic work station
Chromatographic column: Thermo BDS-C18 (250mm * 4.6mm, 5 μ m)
Phase flows: methyl alcohol: water=55: 45 (volume ratio)
Detected temperatures: 30 ℃
Detecting device: UV-detector
Detect wavelength: 306nm
Vacuum pump: SHB-Ш circulation ability of swimming vacuum pump
Electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd. provides, and specification is 100,000/
The preparation of the phase that flows: flow according to acetonitrile: water=52: 48 (volume ratio) mixes, and 0.2 μ m aperture filtrator filters, the degassing of SCQ-250W ultrasonic echography.
Adopt external standard method, need respectively standard items and testing sample solution to be measured.
The standard solution preparation: precision takes by weighing the 50mg standard items, place the brown volumetric flask of 50ml, add and be settled to scale mark with mobile phase dilution again after the ultrasonic 5seconds of small amount of methanol dissolves sample fully, as storing solution, precision is measured storing solution 1ml again, place the brown volumetric flask of 10ml, adding flows is settled to scale mutually.
The testing sample preparation: precision takes by weighing the testing sample with the standard items equivalent weight, places the brown volumetric flask of 50ml, adds to be settled to scale mark with mobile phase dilution again after the ultrasonic 5seconds of small amount of methanol dissolves sample fully.Precision is measured 1ml again, and solution places the brown volumetric flask of 10ml, and adding flows is settled to scale mutually.
According to the cubage formula:
Bring data into and can obtain Resveratrol content.
Above standard items and testing sample are all prepared 3 parts, and absolute error is no more than 0.2%.
The efficient liquid-phase chromatography method-4 that Resveratrol content is measured
Chromatographic condition: adopt U.S. Agilent-1260 high performance liquid chromatograph, Chemstation Edition chromatographic work station
Chromatographic column: Agilent C18 (250mm * 4.6mm, 5 μ m)
Phase flows: methyl alcohol: water=55: 45 (volume ratio)
Detected temperatures: 30 ℃
Detecting device: diode array detector
Detect wavelength: 306nm
Vacuum pump: SHB-Ш circulation ability of swimming vacuum pump
Electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd. provides, and specification is 100,000/
The preparation of the phase that flows: flow according to methyl alcohol: water=55: 45 (volume ratio) mixes, and 0.2 μ m aperture filtrator filters, the degassing of SCQ-250W ultrasonic echography.
Adopt external standard method, need respectively standard items and testing sample solution to be measured.
The standard solution preparation: precision takes by weighing 50mg resveratrol standard items, place the brown volumetric flask of 50ml, add and be settled to scale mark with mobile phase dilution again after the ultrasonic 5seconds of small amount of methanol dissolves sample fully, as storing solution, precision is measured storing solution 1ml again, place the brown volumetric flask of 10ml, adding flows is settled to scale mutually.
The testing sample preparation: precision takes by weighing the testing sample with the standard items equivalent weight, places the brown volumetric flask of 50ml, adds to be settled to scale mark with mobile phase dilution again after the ultrasonic 5seconds of small amount of methanol dissolves sample fully.Precision is measured 1ml again, and solution places the brown volumetric flask of 10ml, and adding flows is settled to scale mutually.
According to the cubage formula:
Bring data into and can obtain Resveratrol content.
Above standard items and testing sample are all prepared 3 parts, and absolute error is no more than 0.2%.
Methodological study of the present invention:
(1) linear relationship is investigated
Under selected optimum determining condition, according to method preparation resveratrol typical curve described above, after the preparation of resveratrol standard solution is finished, behind 0.2 μ m filtering with microporous membrane, inject liquid chromatograph, difference sample introduction 0.2 μ l, 0.5 μ l, 1 μ l, 2 μ l, 3 μ l, 4 μ l, 5 μ l under condition as described below, data regression is carried out regression equation processing, r=1 as a result to sample size and peak area.The result shows that resveratrol has good linear relationship in 0.2～5 μ l scope.Wherein, chromatographic column: Thermo-BDS C18 (250mm * 4.6mm, 5 μ m); Phase flows: methyl alcohol: water (55: 45 volume ratios); Detected temperatures: 30 ℃; Sample size: 5 μ l; Flow velocity: 0.8ml/min; Detecting device: UV-detector; Detect wavelength: 306nm.
(2) range of linearity is investigated
Precision takes by weighing resveratrol standard items 50mg, and phased soln dilution constant volume is made the storing solution that concentration is 1mg/ml with flowing.Adding flows is diluted to the standard solution of 10 μ g/ml, 25 μ g/ml, 50 μ g/ml, 80 μ g/ml, 100 μ g/ml, 120 μ g/ml, 150 μ g/ml and 200 μ g/ml mutually respectively, according to analytical approach of the present invention, sample concentration and peak area are carried out data regression, regression equation is: Y (resveratrol standard items peak area)=36.793X-220.41, r=1 (n=6) (X represents standard items concentration) (seeing accompanying drawing 3 for details).The result shows that resveratrol is in 10 μ g/ml～200 μ g/ml concentration ranges, and peak area and resveratrol concentration are good linear relationship, and the minimum sample size of resveratrol can reach 0.05 μ g as calculated.
Wherein, chromatographic column: Thermo-BDS C18 (250mm * 4.6mm, 5 μ m); Phase flows: methyl alcohol: water=55: 45; Detected temperatures: 30 ℃; Sample size 5 μ l; Flow velocity: 0.8ml/min; Detecting device: UV-detector; Detect wavelength: 306nm.
(3) precision is investigated
Precision refers under the test condition of regulation, same even sample, and through the degree of closeness between the sampling and measuring gained result repeatedly, in order to the reappearance of measured value to be described, precision is represented with relative standard deviation (RSD).The present invention is through 6 revision tests, and the gained peak area value sees Table 1, and peak area RSD value is 0.02%.
Table 1 resveratrol standard specimen is the sample introduction peak area value repeatedly
The result shows that this HPLC method detects Resveratrol content precision height, and reappearance is very good.
Wherein, chromatographic column: Thermo-BDS C18 (250mm * 4.6mm, 5 μ m); Phase flows: methyl alcohol: water=55: 45; Detected temperatures: 30 ℃; Sample size 5 μ l; Flow velocity: 0.8ml/min; Detecting device: UV-detector; Detect wavelength: 306nm.
(4) resveratrol stability experiment
Stability under different standing times of resveratrol and the temperature conditions: it is an amount of to get the resveratrol reference substance, be placed in the brown volumetric flask of 10ml, add methyl alcohol dissolving and dilution and make the solution that contains resveratrol 0.1mg among every 1ml, place respectively at normal temperatures 0,2,4,6,8,16,24 and 48h after, utilize UV-detector to measure the size of 306nm place light absorption value.The sample of getting same concentration again takes out behind the heated sealed 20min in 30,40,50,60,70 and 80 ℃ thermostat water bath respectively, adds methanol constant volume again to scale, surveys the variation of 306nm place light absorption value.(see figure 4) shows indoor at no direct light photograph as a result, and normal temperature is placed 24h, and is little to the content influence of resveratrol.If surpass 48h, part can take place decompose, so prepare resveratrol lucifuge as far as possible.
About the stability of resveratrol to heat, in 30～60 ℃ of scopes, the 20min that heats, absorbance changes little, namely little to the influence of resveratrol, but when higher temperature, as more than 60 ℃, heat behind the 20min, light absorption value can descend (seeing Fig. 5 for details), and resveratrol part takes place decomposes.So extraction, purifying, preservation process are carried out as far as possible at a lower temperature.
Claims (5)
1. high performance liquid chromatography (HPLC) method that Resveratrol content is measured in the giant knotweed, it is characterized in that flowing is the mixed solution of methyl alcohol and water mutually, wherein select the volume fraction of methyl alcohol to be respectively 80%, 65% and 50%, the volume fraction of water is respectively 20%, 35% and 50%, optimize the best methyl alcohol volume fraction 55% of peak time and peak type at last, the mobile phase that water volume mark 45% detects as system.
2. the method for claim 1 is characterized in that chromatographic column is is the reverse-phase chromatographic column of filling material with the octadecylsilane chemically bonded silica.
3. the method for claim 1 is used UV-detector, and the detection wavelength is 306nm, and 30 ℃ of column temperatures, flow velocity are 0.8ml/min, and sampling volume is 5 μ l.
4. the method for claim 1 is characterized in that the sample solution concentration of preparing is 1mg/ml.
5. the method for claim 1 is characterized in that this HPLC method adopts external standard method to carry out content of resveratrol, and concentration range is 6 μ g～200 μ g/ml in concentration and the peak area linear relationship.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290604A (en) * | 2016-07-21 | 2017-01-04 | 林悦安 | Resveratrol evaluates method and the detection thereof of antioxidant radical metabolism |
| CN109709226A (en) * | 2018-12-29 | 2019-05-03 | 中国农业科学院油料作物研究所 | A method of trans-resveratrol content in measurement peanut dry seeds |
| CN113866292A (en) * | 2021-09-13 | 2021-12-31 | 河北威远生物化工有限公司 | Method for measuring content of o-chlorobenzyl magnesium chloride |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290604A (en) * | 2016-07-21 | 2017-01-04 | 林悦安 | Resveratrol evaluates method and the detection thereof of antioxidant radical metabolism |
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| CN109709226A (en) * | 2018-12-29 | 2019-05-03 | 中国农业科学院油料作物研究所 | A method of trans-resveratrol content in measurement peanut dry seeds |
| CN113866292A (en) * | 2021-09-13 | 2021-12-31 | 河北威远生物化工有限公司 | Method for measuring content of o-chlorobenzyl magnesium chloride |
| CN113866292B (en) * | 2021-09-13 | 2024-03-19 | 河北威远生物化工有限公司 | Method for measuring content of o-chlorobenzyl magnesium chloride |
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Application publication date: 20130807 |