CN107271581B - A method of efficiently measuring phenolic compound in citrusfruit using HPLC - Google Patents
A method of efficiently measuring phenolic compound in citrusfruit using HPLC Download PDFInfo
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- CN107271581B CN107271581B CN201710325670.4A CN201710325670A CN107271581B CN 107271581 B CN107271581 B CN 107271581B CN 201710325670 A CN201710325670 A CN 201710325670A CN 107271581 B CN107271581 B CN 107271581B
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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
- G01N30/14—Preparation by elimination of some components
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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
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
Abstract
The present invention provides a kind of methods that phenolic compound in citrusfruit is efficiently measured using HPLC, this method comprises the following steps: (1) after grinding citrusfruit, it is placed in extracting solution and carries out ultrasound assisted extraction, film filtering is carried out after centrifugation, takes supernatant as sample；The extracting solution by methanol, water and formic acid by volume 70: 28: 2 ratio form；(2) sample obtained by step (1) is detected using high performance liquid chromatograph.For the present invention when extracting to citrus, extracting method is simple, without cumbersome extraction step；10 kinds of phenolic substancess in detection citrus while the present invention can be accurate；Detection time of the present invention is short, the excellent effect in terms of detection limit, precision and the rate of recovery.
Description
Technical field
The invention belongs to chemical analysis fields, and in particular to a kind of efficiently to measure phenols chemical combination in citrusfruit using HPLC
The method of object.
Background technique
Phenolic substances has a variety of effects such as anti-inflammatory anti-oxidant, antiallergy, anticancer, antimutation, antibacterial, at present in fruit
The research report of phenolic substances measurement is more.
In numerous detection methods, high performance liquid chromatography is more common, has the advantage that analysis speed is fast, divides
From high-efficient, detection sensitivity is high, detects automation, applied widely etc..
Currently, it is more with the correlative study of aldehydes matter content in HPLC method measurement citrusfruit, but existing research is being surveyed
Determine in citrus phenolic substances there is also sample extraction complex steps, solvent consumption is big, disengaging time is long, separate substance type is few
The deficiencies of.As the method recorded in " content of 18 kinds of flavonoids in hplc simultaneous determination citrusfruit " needs time-consuming
60 minutes, and need to expend a large amount of reagent at the extraction and extraction step is cumbersome；For another example " Phenolic compounds
Characterization and biological activities of Citrus aurantium bloom " in method
Though detectable 6 kinds of phenolic acid and 6 kinds of flavonoid substances, the detection time that respectively need to expend 60 minutes；For another example Chinese patent " utilizes
The method of 17 kinds of phenolic substancess in HPLC measurement grape and citrusfruit " there is also need to carry out cumbersome extraction, dense at the extraction
Contracting and the disadvantage for being redissolved step and detection time length.
Importantly, prior art comparative maturity in terms of individually surveying citrus flavonoids or phenolic acid, but to flavonoids and
Phenol acid substance measures aspect simultaneously and there is problems: detection substance is few, sample treatment is cumbersome, detection time is longer etc..Such as
Existing research report, is not detected aurantiin, neohesperidin, shaddock when detecting Navel Orange Fruits kind flavonoids with ultra high efficiency liquid phase
Pi Susan kind flavanone phenolic substances.For another example, when carrying out sample treatment, the prior art often carry out freeze-drying or
Prolonged concussion is extracted.
In conclusion this field needs a kind of method that the HPLC being simple and efficient detects phenolic compound in citrus.
Summary of the invention
In view of the shortcomings of the prior art, it is efficiently measured in citrusfruit the purpose of the present invention is to provide a kind of using HPLC
The method of phenolic compound, this method comprises the following steps:
(1) it after grinding citrusfruit, is placed in extracting solution and carries out ultrasound assisted extraction, film filtering is carried out after centrifugation, is taken
Supernatant is as sample；The extracting solution by methanol, water and formic acid by volume 70: 28: 2 ratio form；
(2) sample obtained by step (1) is detected using high performance liquid chromatograph, chromatographic condition are as follows:
Chromatographic column C18:5um, 4.6mm × 150mm；Mobile phase A is 10% formic acid, and Mobile phase B is acetonitrile；Flow velocity:
0.8ml/min；30 DEG C of column temperature；Gradient elution: 0~25min, 95% mobile phase A, the mobile phase A of 5% Mobile phase B -85%,
15% Mobile phase B；25~42min, 85% mobile phase A, the mobile phase A of 15% Mobile phase B -78%, 22% Mobile phase B；42~
50min, 78% mobile phase A, the mobile phase A of 22% Mobile phase B -64%, 36% Mobile phase B；50~55min, 64% mobile phase A,
The mobile phase A of 36% Mobile phase B -95%, 5% Mobile phase B.
The phenolic compound is gallic acid, P-hydroxybenzoic acid, caffeic acid, sinapic acid, chlorogenic acid, ferulic acid, reed
Fourth, aurantiin, naringenin and neohesperidin.
Preferably, in step (1), before grinding, citrus is placed in ultra low temperature freezer and is saved, storage temperature is -80 DEG C.
Preferably, in step (1), the w/v (g/ml) of citrusfruit and extracting solution is 1:3.
Preferably, in step (1), ultrasonic power is (5000r/min), and the ultrasound assisted extraction time is 30 minutes.
Preferably, in step (1), when carrying out film filtering, using 0.45 μm of organic filter membrane of micropore.
Optionally, in step (2), high performance liquid chromatograph used is 1260 highly effective liquid phase chromatographic system of Agilent.
In conjunction with the experimental data of the embodiment of the present invention and the report of the prior art, it is known that this method biggest advantage
While being to guarantee detection phenolic substances diversification, sample pre-processing is simple, easy to operate, the sample of such as many research reports
Product pretreatment, which needs to be freeze-dried or shake, extracts 12h etc..This method is reproducible (RSD is less than 3.52%) simultaneously, accurate
It spends high (0.38%~1.90%), sample recovery rate height (90.26%~118.85%), therefore can effectively detect in citrus
Phenolic substances, if existing research report, with ultra high efficiency liquid phase detect Navel Orange Fruits kind flavonoids when be not detected aurantiin,
Three kinds of neohesperidin, naringenin flavanone phenolic substancess, and this method detection limit is lower than 0.03 μ gg-1, can be in pulp and fruit
This 3 kinds of flavanones are detected in skin.Therefore this method can it is more efficient using phenolic substances in citrus in terms of have certain reference
Value.
Beneficial effects of the present invention:
(1) for the present invention when extracting to citrus, extracting method is simple, without cumbersome extraction step；
(2) 10 kinds of phenolic substancess in detection citrus while the present invention can be accurate；
(3) detection time of the present invention is short, the excellent effect in terms of detection limit, precision and the rate of recovery.
Detailed description of the invention
Fig. 1 is the HPLC figure of 9 kinds of phenolic compound mixed standard solutions；Fig. 1 is to exist respectively in hybrid standard product 50min
Chromatogram under 280nm, 320nm, 367nm, 283nm wavelength, initial wavelength are 280nm, are switched in 4min
320nm is switched to 367nm in 23min, is switched to 283nm in 26min；According to the difference of retention time, peak is followed successively by not
Gallate-based, chlorogenic acid, caffeic acid, ferulic acid, sinapic acid, rutin, aurantiin, neohesperidin, naringenin；
Fig. 2 is that the HPLC of P-hydroxybenzoic acid standard solution schemes；Fig. 2 is P-hydroxybenzoic acid standard items in 260nm wavelength
Under chromatogram.
Fig. 3 is that phenolic compound first time wavelength (predominantly detects caffeic acid, mustard in " blood orange " kind Meat Sample
HPLC acid, chlorogenic acid, ferulic acid, gallic acid, aurantiin, neohesperidin, naringenin, similarly hereinafter) schemes, wherein 1. galla turcicas
Acid；2. chlorogenic acid；3. caffeic acid；4. sinapic acid；5. aurantiin；
Fig. 4 is that second of wavelength of phenolic compound (predominantly detects rutin, to hydroxyl in " blood orange " kind Meat Sample
HPLC figure yl benzoic acid, similarly hereinafter), wherein 1. P-hydroxybenzoic acid；2. rutin；
Fig. 5 is the HPLC figure of phenolic compound first time wavelength in " blood orange " kind peel sample, wherein 1. do not have
Gallate-based；2. chlorogenic acid；3. caffeic acid；4. ferulic acid；5. sinapic acid；6. aurantiin；7. neohesperidin；8. naringenin；
Fig. 6 is the HPLC figure of second of wavelength of phenolic compound in " blood orange " kind peel sample, wherein 1. pairs
Hydroxybenzoic acid；2. rutin；
Fig. 7 is the HPLC figure of phenolic compound first time wavelength in " fertile mandarin orange " kind Meat Sample, wherein 1. gallic acids；
2. chlorogenic acid；3. caffeic acid；4. sinapic acid；5. aurantiin；
Fig. 8 is the HPLC figure of second of wavelength of phenolic compound in " fertile mandarin orange " kind Meat Sample, wherein 1. para hydroxybenzenes
Formic acid；2. rutin；
Fig. 9 is the HPLC figure of phenolic compound first time wavelength in " fertile mandarin orange " kind peel sample, wherein 1. gallic acids；
2. chlorogenic acid；3. caffeic acid；4. ferulic acid；5. sinapic acid；6. aurantiin；7. neohesperidin；8. naringenin；
Figure 10 is the HPLC figure of second of wavelength of phenolic compound in " fertile mandarin orange " kind peel sample, wherein 1. para hydroxybenzenes
Formic acid；2. rutin.
Specific embodiment
The present invention is specifically described below by embodiment, it is necessary to which indicated herein is that following embodiment is only used
It is further detailed in the present invention, should not be understood as limiting the scope of the invention, which is skilled in technique
Some nonessential modifications and adaptations that personnel are made according to foregoing invention content, still fall within protection scope of the present invention.
Embodiment 1
Reagent and instrument:
Standard items: naringenin (Naringenin), aurantiin (Naringin), neohesperidin (Neohesperidin), coffee
Coffee acid (Caffeic acid), sinapic acid (Sinapic acid), chlorogenic acid (Chlorogenic acid), ferulic acid
(Ferulic acid), gallic acid (Gallic acid), rutin (Rutin) and P-hydroxybenzoic acid (p-
Hydroxybenzoic acid), it is purchased from Sigma company, purity is 98% or more.Methanol, formic acid and acetonitrile are chromatography
It is pure, it is purchased from Mike woods company；Water is laboratory Mi Libo ultrapure water.
Supersonic wave cleaning machine, centrifuge, 1260 highly effective liquid phase chromatographic system of Agilent, C18 chromatographic column: 4.6mm ×
150mm, 5um.
1, the drafting of standard curve: respectively being dissolved every bottle of quantitative 20mg standard items with Chromatographic Pure Methanol, constant volume in
In 10mL brown volumetric flask, it is configured to the standard solution of 2mg/mL.Whole process is all protected from light, and above-mentioned mother liquor is flowed
It is mutually configured to the concentration gradient of 0.25,0. 5,1,2,5,10,25,50,100mg/L respectively, then is configured to mixed standard solution,
It is kept in dark place in -20 DEG C of environment below.Chromatography is carried out under chromatographic condition, and calculates the recurrence of each standard items
Equation and related coefficient.
2, in sample phenolic substances extraction: take fruit into mortar from -80 DEG C of refrigerators, accurately weigh 0.5g aggregate sample
It is put into 2mL centrifuge tube, 1.5mL extracting solution is added under the conditions of being protected from light and (methanol: water: formic acid=70: 28: 2), mixes well.It will
Centrifuge tube is put into assisted extraction in Vltrasonic device, after ultrasonic 30min, is centrifuged 15min under 4 DEG C, 5000r/min centrifugal condition.
With 0.45 μm of filtering with microporous membrane supernatant, it is stored in -20 DEG C of analyses for phenolic acid and flavonoids.
3, efficient liquid phase chromatographic analysis is carried out to sample:
Chromatographic condition:
Use 1260 liquid chromatograph of Agilent, C18 chromatographic column: 5um, 4.6mm × 150mm；30 DEG C of column temperature；Sample volume
20uL；Mobile phase: A is 10% formic acid solution (volume ratio), and B is acetonitrile, gradient elution, shown in elution program table 1；Flow velocity is
0.8mL/min。
1 proportion of mobile phase of table and elution time
Table 3 Flow matching ratio and elution time
Aurantiin, neohesperidin, naringenin 283nm wavelength detecting；Caffeic acid, sinapic acid, chlorogenic acid, ferulic acid are used
320nm wavelength detecting；P-hydroxybenzoic acid 260nm wavelength detecting；Gallic acid 280nm wavelength detecting；Rutin 367nm
Wavelength detecting.
Wavelength can only be arranged in detector due to testing 1260 instrument of Agilent used in method, and cannot detect automatically
And it is adjusted to optimum wavelength and is measured.For that can detect automatically and adjust the instrument of optimum wavelength, it may not be necessary to carry out hand
Work setting.
Rutin and sinapic acid appearance time are close, and P-hydroxybenzoic acid and chlorogenic acid appearance time are close, in the sample without
The time of wavelength switching is accurately arranged in method, that is, twice, detecting 0min setting wavelength for the first time is for a sample measurement when detecting
280nm, 4min are converted to 320nm, and 25min converts 283nm, and caffeic acid, sinapic acid, green can be detected under this wavelength condition
Ortho acid, ferulic acid, aurantiin, neohesperidin, naringenin, gallic acid；0min setting wavelength is 260nm when second of detection,
20min is converted to 367nm, and P-hydroxybenzoic acid, rutin can be verified under this wavelength condition.
4, by the analysis result of sample solution compared with standard curve, the phenolic compound calculated in sample solution contains
Amount.
Standard curve equation of linear regression, related coefficient, minimum detection limit, the result of relative standard deviation and the rate of recovery are such as
Shown in table 2.
Table 2
Claims (6)
1. a kind of method for efficiently measuring phenolic compound in citrusfruit using HPLC, which is characterized in that the method includes
Following steps:
(1) it after grinding citrusfruit, is placed in extracting solution and carries out ultrasound assisted extraction, film filtering is carried out after centrifugation, takes supernatant
Liquid is as sample；The extracting solution by methanol, water and formic acid by volume 70: 28: 2 ratio form；
(2) sample obtained by step (1) is detected using high performance liquid chromatograph, chromatographic condition are as follows:
C18:5 μm of chromatographic column, 4 .6mm × 150mm；Mobile phase A is 10% formic acid, and Mobile phase B is acetonitrile；Flow velocity: 0 .8ml/
min；30 DEG C of column temperature；Gradient elution: 0~25min, 95 % mobile phase As, the mobile phase A of 5% Mobile phase B -85%, 15% Mobile phase B；
25~42min, 85% mobile phase A, the mobile phase A of 15% Mobile phase B -78%, 22% Mobile phase B；42~50min, 78% mobile phase A,
The mobile phase A of 22% Mobile phase B -64%, 36% Mobile phase B；50~55min, 64% mobile phase A, the mobile phase of 36% Mobile phase B -95%
A, 5% Mobile phase B；
The phenolic compound is gallic acid, P-hydroxybenzoic acid, caffeic acid, sinapic acid, chlorogenic acid, ferulic acid, rutin, shaddock
Skin glycosides, naringenin and neohesperidin.
2. the method according to claim 1, wherein before grinding, citrus is placed in ultralow temperature ice in step (1)
It is saved in case, storage temperature is -80 DEG C.
3. the method according to claim 1, wherein in step (1), the bulking value of citrusfruit and extracting solution
Than for 1g:3ml.
4. the method according to claim 1, wherein ultrasonic power is 5000 r/min, ultrasound in step (1)
The assisted extraction time is 30 minutes.
5. the method according to claim 1, wherein in step (1), when carrying out film filtering, using 0 .45 μm it is micro-
The organic filter membrane in hole.
6. the method according to claim 1, wherein high performance liquid chromatograph used is Agilent in step (2)
1260 highly effective liquid phase chromatographic systems.
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| CN108490094B (en) * | 2018-03-30 | 2020-10-02 | 福建省农业科学院农业工程技术研究所 | Method for simultaneously determining 22 flavone and phenolic acid substances in citrus fruits |
| CN108680660A (en) * | 2018-04-03 | 2018-10-19 | 四川农业大学 | A method of polyphenols is extracted and identified from kiwifruit fruit |
| CN110927274B (en) * | 2019-12-03 | 2022-03-15 | 健民药业集团股份有限公司 | Method for simultaneously detecting contents of six ingredients in shitong capsule |
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| CN102706980B (en) * | 2012-06-06 | 2014-01-01 | 福建省农业科学院农业工程技术研究所 | Method utilizing HPLC to test seventeen phenol substances contained in grapes and oranges |
| CN102836179A (en) * | 2012-09-19 | 2012-12-26 | 四川农业大学 | Method for extracting polyphenol material in plants |
| CN103336069B (en) * | 2013-06-13 | 2015-06-10 | 江苏省农业科学院 | HPLC (High Performance Liquid Chromatography) determination method of phenolic compounds in peach fruit |
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