CN103267821A - HPLC-ELSD (high performance liquid chromatography-evaporation light-scattering detection) measuring method of grape berry sugar - Google Patents
HPLC-ELSD (high performance liquid chromatography-evaporation light-scattering detection) measuring method of grape berry sugar Download PDFInfo
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Abstract
The invention discloses an HPLC-ELSD (high performance liquid chromatography-evaporation light-scattering detection) measuring method of grape berry sugar, belonging to the technical field of food sugar detection. The method comprises the following steps: firstly, juicing and homogenizing grapes, ultrasonic extracting the juice, and then, centrifuging the juice, taking the supernatant, filtering the supernatant and using the filtrate as a test sample; respectively preparing standard working solutions with a series of concentrations of glucose, fructose and sucrose; the chromatographic conditions are as follows: a polystyrene-divinyl benzene bonded amino sugar column serves as a chromatographic column, the mobile phase is a mixture of acetonitrile and water with volume ratio of 70-80:30-20, and the flow velocity is 1mL/min, the column temperature is 30-35 DEG C, the detector is ELSD, the temperature of a detecting pool is 40-80 DEG C, and the sample size is 10 microliters; and then inspecting linear dependence and precision, and detecting stability and sample recovery. The method provided by the invention is simple and convenient to operate, small in time consumption, good in measurement result repeatability, high in precision and capable of accurately and quickly measuring the contents of glucose, fructose and sucrose in grape berry.
Description
Technical field
The invention belongs to food sugar detection technique field, particularly a kind of grape fruit sugar HPLC-ELSD assay method.
Background technology
Grape is one of most important fruit in the world.The thin succulence of grape fruit skin, sour-sweet delicious, nutritious.Sugar content can reach 10%～30% in the ripe berry, and the sugar of grape fruit contains a spot of sucrose in addition based on glucose, fructose, is directly absorbed by human body easily; Multiple tartaric acid in the grape fruit helps digestion; The composition such as anthocyanin, flavonoids, resveratrol that contains multiple mineral matter to the human body beneficial, vitamin, amino acid, lecithin and beautifying face and moistering lotion in the grape fruit.Grape is of many uses, can be used for eating raw, wine processed, juice processed and system do.The grape fruit sugar content is the important indicator that influences berry quality.Sugar composition and content height thereof not only directly influence nutritional quality and the local flavor of ripe berry; And as the synthetic precursor substance of anthocyanin, the fruit sugar is formed and content can influence the painted of grape berry, thereby influences the exterior quality of Table Grape and the processing quality of vinifera.Therefore, in the grape fruit growth and development process and maturation when gathering, the sugar of measuring grape accurately, fast is extremely important.
At present, be usually used in separating and the method for measuring saccharic composition has gas chromatography (GC) method and high performance liquid chromatography (HPLC) method etc.But the GC method needs sample to be become gasifiable volatile ingredient at column front derivation, complex operation, length consuming time.H PLC method is simple and convenient than vapor-phase chromatography, need not derivatization, and measure monose, the compound sugar effect is better.The detecting device that the HPLC method detects carbohydrate has differential detecting device (RID), UV-detector, fluorescence detector, electrochemical detector and evaporative light-scattering detector (ELSD) etc.RID is the detecting device commonly used that the HPLC method detects carbohydrate, based on METHOD FOR CONTINUOUS DETERMINATION chromatographic column effluent change of refractive; It to temperature extremely sensitivity make baseline very unstable, incompatible with gradient elution, and detection sensitivity is very low.The light scattering electric signal that ELSD is based on the involatile particles of solute after the atomizing of chromatographic column effluent carries out qualitative, quantitative to sample, its response does not rely on the optical characteristics of sample, do not require that sample has chromophore or fluorophor, thereby have advantages such as good stability, the interference of highly sensitive, solvent-free peak.During tradition high effective liquid chromatography for measuring sugar, the nh 2 column in the chromatographic column commonly used, to acetonitrile purity requirement height, poor stability, fixing easily loss mutually, the life-span is short, and is expensive.The target peak of ODS post (the octadecylsilane chemically bonded silica filler also claims the C18 post) separates not exclusively, and effect is undesirable.The efficient sugared post of Waters, the efficient sugared post of Zorbaxa etc. are isocratic elution, and if the separation of some sugar good not as gradient elution is gradient elution then may cause the chemical degradation of its post bed and influence post and imitate.Other ion-type sugar post then must just can make some sugared compositions reach separation under high column temperature.Prevail sugar post is as the sugared post of special use, amino sugar post for the polystyrene-divinylbenzene bonding, the problem that does not have other sugared post of puzzlement such as nh 2 column, efficient sugared post, ODS post and other ion-types sugar post, there is not the chemical degradation phenomenon, good separation, and also very stable under the situation of room temperature and gradient elution.
Summary of the invention
The detection method that the purpose of this invention is to provide a kind of grape fruit sugar, this method utilize the HPLC-ELSD assay method to detect the grape fruit sugar.
A kind of grape fruit sugar HPLC-ELSD assay method comprises the steps:
1) sampling
Grape is thanked and is spent back 20-25d to rise, and in three positions of East, West, South, North four direction and upper, middle and lower of plant, takes the fruit of median size, no disease and pest, adopts sample one time every 7-10d; Put into ice chest after fruit is adopted rapidly, take back the laboratory and put into-80 ℃ of refrigerators standby;
2) sample preparation
Get respectively size evenly, 15～20 of consistent clean grapes that dries of color and luster, smash and mixing with juice extractor, accurately take by weighing homogenate 1.00g rapidly, the ethanol constant volume with 90% is weighed in the 25mL volumetric flask; At normal temperatures, extract 20-40min with ultrasound wave, take out, after the room temperature cooling, add 90% ethanol add to before the weight of liquid during constant volume 25mL; To extract the centrifugal 15-30min of 8000-12000r/min under the sample normal temperature, get supernatant, behind 0.25 μ m membrane filtration, be used as for test agent;
3) preparation of standard solution
Be respectively the series concentration standard operation liquid of glucose, fructose and the sucrose of 0.1mg/mL, 0.3mg/mL, 0.6mg/mL, 0.9mg/mL, 1.2mg/mL, 1.5mg/mL with the ultrapure water compound concentration; Through 0.25 μ m membrane filtration, get the filtrate sample introduction before the sample introduction;
4) chromatographic condition
Chromatographic column is the amino sugar post Prevail carbohydrate ES(250mm * 4.6mm of polystyrene-divinylbenzene bonding, 5 μ m); Flowing is the potpourri of acetonitrile and water mutually, and wherein, the volume ratio of acetonitrile and water is 70-80:30-20; Flow rate of mobile phase is 1mL/min; Column temperature: 30 ℃-35 ℃; Detecting device is selected evaporative light-scattering detector (ELSD) for use, 40 ℃-80 ℃ of detection cell temperature, sample size 10 μ L;
5) linear dependence and precision are investigated
Series standard working fluid with the glucose of 0.1mg/mL, the 0.3mg/mL of step 3) preparation, 0.6mg/mL, 0.9mg/mL, 1.2mg/mL, 1.5mg/mL, fructose, sucrose, set by step 4) described chromatographic condition sample introduction is analyzed and is calculated, and gets the typical curve regression equation;
Draw same sample solution, repeat sample introduction 3-7 time, set by step 4) described chromatographic condition sample introduction analysis, with fructose, dextrose plus saccharose calculated by peak area precision;
6) Detection of Stability
Draw same sample solution, respectively at seasoning under the room temperature 0,2,4,6,8,16 and 24h after, set by step 4) described chromatographic condition sample introduction analyzes, and repeats sample introduction 3-7 time; Test fructose, dextrose plus saccharose peak area also calculates its relative standard deviation (RSD);
7) sample recovery rate detects
In many parts that prepare same grape sample extracting solutions, add an amount of glucose, fructose, sucrose standard specimen, other has 1 part of grape sample extracting solution not add glucose, fructose, sucrose standard specimen; Set by step 2) described sample treatment carries out pre-service, set by step 4) described chromatographic condition sample introduction is analyzed, and continuous sample introduction 5 times calculates its recovery; According to said method measure the recovery height of fructose, glucose, sucrose;
8) key instrument and reagent
U.S. Agilent1260 high performance liquid chromatograph, the Alltech2000ES evaporative light-scattering detector; Fructose, glucose sugar, sucrose, acetonitrile are chromatographically pure, and water is ultrapure water.
Beneficial effect of the present invention is:
The present invention utilizes the sugar in high performance liquid chromatography-evaporative light-scattering detection method (HPLC-ELSD) mensuration grape fruit, at first in grape fruit sugar leaching process, adopts ethanol and ultrasound wave to handle and combines, and easy and simple to handle, sugar extracts fully, weak point consuming time.Simultaneously, when measuring fruit glucose, fructose, cane sugar content, adopt Prevail to combine with HPLC and ELSD, both can use the gradient phase that flows, reduce disengaging time, improve separating effect, can guarantee good stability under the room temperature again, and improve the sensitivity of detecting device; The measurement result good reproducibility, the precision height.The invention provides a kind of method that can measure fructose in the grape fruit, glucose, cane sugar content simply, accurately, fast simultaneously, practical, can be used for the accurate detection of fruit sugar.
Description of drawings
Fig. 1 is that concentration is that the high-efficient liquid phase color of 0.6mg/mL sucrose standard sample is composed peak figure in the embodiment of the invention 1.
Fig. 2 is that concentration is that the high-efficient liquid phase color of 0.6mg/mL glucose standard sample is composed peak figure in the embodiment of the invention 1.
Fig. 3 is that concentration is that the high-efficient liquid phase color of 0.6mg/mL fructose standard sample is composed peak figure in the embodiment of the invention 1.
Embodiment
The present invention will be further described in detail below in conjunction with accompanying drawing and specific embodiment:
Key instrument and reagent that following embodiment uses are as follows:
U.S. Agilent1260 high performance liquid chromatograph, the Alltech2000ES evaporative light-scattering detector; Fructose, glucose sugar, sucrose, acetonitrile are chromatographically pure, and water is ultrapure water.
Embodiment 1:
(1) material
Pengshan County, Meishan City, Sichuan Province take shelter from rain early-ripening grape kind ' summer black ', late grape kind ' the red earth ' and ' beauty refers to ' of cultivation and the late grape kind of outdoor cropping ' the red earth '.
(2) method
1) sampling
Grape is thanked and is spent back 20-25d to play that (early-maturing variety ' summer black ' is spent back 20d for thanking sample time, late variety ' the red earth ' and ' beauty refers to ' sample time are spent back 25d for thanking), three positions of East, West, South, North four direction and fruit ear upper, middle and lower plant, take the fruit of median size, no disease and pest, be 7d every 7-10d(early-maturing variety ' summer black ' sampling interval time, late variety ' the red earth ', ' beauty refers to ' sampling interval time are 10d) adopt sample one time; Put into ice chest after fruit is adopted rapidly, take back the laboratory and put into-80 ℃ of refrigerators standby;
2) sample preparation
Get respectively size evenly, 15～20 of consistent clean grapes that dries of color and luster, smash and mixing with juice extractor, accurately take by weighing homogenate 1.00g rapidly, the ethanol constant volume with 90% is weighed in the 25mL volumetric flask; At normal temperatures, extract 20-40min with ultrasound wave, take out, after the room temperature cooling, add 90% ethanol add to before the weight of liquid during constant volume 25mL; To extract the centrifugal 15min of 8000-12000r/min under the sample normal temperature, get supernatant, through 0.25 μ m membrane filtration, do for test agent;
3) preparation of standard solution
Accurately take by weighing 50mg standard glucose, fructose, sucrose, be settled to 10mL with ultrapure water respectively, be mixed with the standard solution that concentration is 5mg/mL; And then be mixed with the series standard working fluid that concentration is 0.1mg/mL, 0.3mg/mL, 0.6mg/mL, 0.9mg/mL, 1.2mg/mL, 1.5mg/mL; Through 0.25 μ m membrane filtration, get the filtrate sample introduction before the sample introduction;
4) chromatographic condition
Chromatographic column is the amino sugar post Prevail carbohydrate ES(250mm * 4.6mm of polystyrene-divinylbenzene bonding, 5 μ m) (Prevail sugar post does not exist other sugared post of puzzlement such as nh 2 column, efficient sugared post and other ion-type sugar post C as the sugared post of special use
18Problem, do not have the chemical degradation phenomenon, good separation, and also very stable under the situation of room temperature and gradient elution); Flow be mutually acetonitrile-water (78:22, V:V), flow velocity: 1mL/min; Column temperature: 35 ℃; Detecting device selects for use evaporative light-scattering detector (ELSD) (with advantages such as good stability, the interference of highly sensitive, solvent-free peak, remedied the variation that traditional RID is subjected to temperature, flow velocity, unstability of base line is fixed, and can not be used for the deficiency of gradient elution), 50 ℃ of detection cell temperature; Sample size 10 μ L;
5) linear dependence and precision are investigated
Series standard working fluid with 0.1mg/mL, 0.3mg/mL, 0.6mg/mL, 0.9mg/mL, 1.2mg/mL, 1.5mg/mL glucose, fructose, sucrose, set by step 3) described chromatographic condition sample introduction is analyzed and is calculated, get the typical curve regression equation, related coefficient is 0.9997～0.9998, and linear relationship is good;
Draw same sample solution, repeat sample introduction 6 times, set by step 4) described chromatographic condition sample introduction analyzes, with fructose, dextrose plus saccharose calculated by peak area precision; Measured value is respectively 0.71%, 0.92% and 0.96%, shows that this method precision is good;
6) Detection of Stability
Draw same sample solution, respectively at seasoning under the room temperature 0,2,4,6,8,16 and 24h after, set by step 3) described chromatographic condition sample introduction analyzes, and repeats sample introduction 3 times; Test fructose, dextrose plus saccharose peak area and calculate its relative standard deviation (RSD) and be respectively 1.5%, 2.69% and 1.8%; As seen this method has good stability to the assay of fructose, dextrose plus saccharose in the grape fruit;
7) sample recovery rate detects
7 parts of the same sample grape extracts of absorption known sugars content, wherein 6 parts add glucose, fructose, each 25mg of sucrose standard specimen, 25mg, 2.8mg, and other has 1 part of grape extract not add glucose, fructose, sucrose standard specimen; Set by step 5) described sample treatment carries out pre-service, set by step 3) described chromatographic condition sample introduction is analyzed, and continuous sample introduction 5 times calculates its recovery; According to said method measure the recovery height of fructose, glucose, sucrose, all more than 98%.
8) grape sample sugar is measured
Set by step 5) sample treatment described in carries out the grape fruit The pretreatment, set by step 4) described chromatographic condition sample introduction is analyzed, grape fruit under take shelter from rain cultivation and two kinds of cultivation modes of outdoor cropping is carried out the mensuration of sugar content, the content of fructose, glucose, sucrose in all can fine mensuration fruit, and target peak noiseless (the results are shown in Figure 1, Fig. 2 and Fig. 3).
Claims (6)
1. a grape fruit sugar HPLC-ELSD assay method is characterized in that, comprises the steps:
1) sample preparation
Get big or small even, the consistent clean grape that dries of color and luster, smash and mixing with juice extractor, accurately take by weighing homogenate 1.00g rapidly, the ethanol constant volume with 90% is weighed in the 25mL volumetric flask; Extract 20-40min with ultrasound wave then, take out, after the room temperature cooling, add 90% ethanol add to before the weight of liquid during constant volume 25mL; To extract the centrifugal 15-30min of 8000-12000r/min under the sample normal temperature, get supernatant, behind 0.25 μ m membrane filtration, be used as for test agent;
2) preparation of standard solution
Be respectively the series concentration standard operation liquid of glucose, fructose and the sucrose of 0.1mg/mL, 0.3mg/mL, 0.6mg/mL, 0.9mg/mL, 1.2mg/mL, 1.5mg/mL with the ultrapure water compound concentration;
3) chromatographic condition
Chromatographic column is the amino sugar post of polystyrene-divinylbenzene bonding; Flowing is the potpourri of acetonitrile and water mutually, and wherein, the volume ratio of acetonitrile and water is 70-80:30-20; Flow rate of mobile phase is 1mL/min; Column temperature: 30 ℃-35 ℃; Detecting device is selected evaporative light-scattering detector for use, 40 ℃-80 ℃ of detection cell temperature, sample size 10 μ L;
4) linear dependence and precision are investigated
With step 2) the series standard working fluid of 0.1mg/mL, 0.3mg/mL, 0.6mg/mL, the 0.9mg/mL of preparation, the glucose of 1.2mg/mL, 1.5mg/mL, fructose, sucrose, set by step 3) described chromatographic condition sample introduction is analyzed and is calculated, and gets the typical curve regression equation;
Draw same sample solution, repeat sample introduction 3-7 time, set by step 3) described chromatographic condition sample introduction analysis, with fructose, dextrose plus saccharose calculated by peak area precision;
5) Detection of Stability
Draw same sample solution, respectively at seasoning under the room temperature 0,2,4,6,8,16 and 24h after, set by step 3) described chromatographic condition sample introduction analyzes, and repeats sample introduction 3-7 time; Test fructose, dextrose plus saccharose peak area also calculates its relative standard deviation;
6) sample recovery rate detects
In many parts that prepare same grape sample extracting solutions, add glucose, fructose, sucrose standard specimen, other has 1 part of grape sample extracting solution not add glucose, fructose, sucrose standard specimen; Set by step 1) described sample treatment carries out pre-service, set by step 3) described chromatographic condition sample introduction is analyzed, and continuous sample introduction 5 times calculates its recovery.
2. grape fruit sugar HPLC-ELSD assay method according to claim 1 is characterized in that, gets
Quadrat method is as follows:
Grape is thanked and is spent back 20-25d to rise, and in three positions of East, West, South, North four direction and upper, middle and lower of plant, takes the fruit of median size, no disease and pest, adopts sample one time every 7-10d; Put into ice chest after fruit is adopted rapidly, take back the laboratory and put into-80 ℃ of refrigerators standby.
3. grape fruit sugar HPLC-ELSD assay method according to claim 1 is characterized in that, with step 2) described in standard solution before sample introduction through 0.25 μ m membrane filtration, get the filtrate sample introduction.
4. grape fruit sugar HPLC-ELSD assay method according to claim 1 is characterized in that the volume ratio of the acetonitrile described in the step 3) and water is 78:22.
5. grape fruit sugar HPLC-ELSD assay method according to claim 1 is characterized in that the column temperature described in the step 3) is 35 ℃.
6. grape fruit sugar HPLC-ELSD assay method according to claim 1 is characterized in that 50 ℃ of the detection cell temperature described in the step 3).
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| CN103543222A (en) * | 2013-10-01 | 2014-01-29 | 江苏康缘药业股份有限公司 | Reduning injection saccharide content detection method |
| CN104280472A (en) * | 2014-10-16 | 2015-01-14 | 江苏省农业科学院 | Method for detecting sugar components in fruits by liquid chromatography carried VWD (variable wavelength detector) |
| CN104483431A (en) * | 2014-12-24 | 2015-04-01 | 江苏省农业科学院 | Method for separating and purifying anthocyanin from blood-flesh peaches |
| CN104807943A (en) * | 2015-05-12 | 2015-07-29 | 广西壮族自治区梧州食品药品检验所 | Determination method of glucose content in glucose injection |
| CN104931602A (en) * | 2015-05-12 | 2015-09-23 | 广西壮族自治区梧州食品药品检验所 | Method for determining content of glucose and sodium chloride in sodium chloride and dextrose injection |
| CN105021760A (en) * | 2014-04-30 | 2015-11-04 | 天津药物研究院 | Determination method for content of inverted sugar in inverted sugar electrolyte injection |
| CN105675760A (en) * | 2016-02-01 | 2016-06-15 | 杏辉天力（杭州）药业有限公司 | Method for detecting content of total sugar in Chinese red dates |
| CN107449840A (en) * | 2017-07-13 | 2017-12-08 | 宁波市食品检验检测研究院 | A kind of detection method of formula milk/determination of FOS in milk powder total amount |
| CN111175408A (en) * | 2020-02-12 | 2020-05-19 | 深圳大学 | High performance liquid chromatography method for determining soluble sugar in tomato fruits |
| CN111198241A (en) * | 2020-03-17 | 2020-05-26 | 乐斯福(明光)有限公司 | Sugar content detection method for baking powder used in cold climate |
| CN113075324A (en) * | 2021-03-30 | 2021-07-06 | 西南大学 | Method for determining sugar and organic acid content in loquat fruits by using HPLC (high Performance liquid chromatography) |
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| CN103543222A (en) * | 2013-10-01 | 2014-01-29 | 江苏康缘药业股份有限公司 | Reduning injection saccharide content detection method |
| CN105021760A (en) * | 2014-04-30 | 2015-11-04 | 天津药物研究院 | Determination method for content of inverted sugar in inverted sugar electrolyte injection |
| CN104280472A (en) * | 2014-10-16 | 2015-01-14 | 江苏省农业科学院 | Method for detecting sugar components in fruits by liquid chromatography carried VWD (variable wavelength detector) |
| CN104483431A (en) * | 2014-12-24 | 2015-04-01 | 江苏省农业科学院 | Method for separating and purifying anthocyanin from blood-flesh peaches |
| CN104807943A (en) * | 2015-05-12 | 2015-07-29 | 广西壮族自治区梧州食品药品检验所 | Determination method of glucose content in glucose injection |
| CN104931602A (en) * | 2015-05-12 | 2015-09-23 | 广西壮族自治区梧州食品药品检验所 | Method for determining content of glucose and sodium chloride in sodium chloride and dextrose injection |
| CN104807943B (en) * | 2015-05-12 | 2016-08-24 | 广西壮族自治区梧州食品药品检验所 | A kind of assay method of glucose in injection |
| CN105675760A (en) * | 2016-02-01 | 2016-06-15 | 杏辉天力（杭州）药业有限公司 | Method for detecting content of total sugar in Chinese red dates |
| CN107449840A (en) * | 2017-07-13 | 2017-12-08 | 宁波市食品检验检测研究院 | A kind of detection method of formula milk/determination of FOS in milk powder total amount |
| CN111175408A (en) * | 2020-02-12 | 2020-05-19 | 深圳大学 | High performance liquid chromatography method for determining soluble sugar in tomato fruits |
| CN111198241A (en) * | 2020-03-17 | 2020-05-26 | 乐斯福(明光)有限公司 | Sugar content detection method for baking powder used in cold climate |
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Application publication date: 20130828 |