CN108169385A - A kind of method using six kinds of glucides in ultra performance liquid chromatography concatenation QDa simultaneously quick detection health liquor - Google Patents

A kind of method using six kinds of glucides in ultra performance liquid chromatography concatenation QDa simultaneously quick detection health liquor Download PDF

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Publication number
CN108169385A
CN108169385A CN201810201940.5A CN201810201940A CN108169385A CN 108169385 A CN108169385 A CN 108169385A CN 201810201940 A CN201810201940 A CN 201810201940A CN 108169385 A CN108169385 A CN 108169385A
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Prior art keywords
glucides
kinds
sample
health liquor
solution
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CN201810201940.5A
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CN108169385B (en
Inventor
王银辉
高家坤
崔玉军
刘国英
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Anhui Gujing Distillery Co Ltd
Anhui Ruisiweier Technology Co Ltd
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Anhui Gujing Distillery Co Ltd
Anhui Ruisiweier Technology Co Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/62Detectors specially adapted therefor
    • G01N30/72Mass spectrometers

Abstract

The invention discloses a kind of methods using six kinds of glucides in ultra performance liquid chromatography concatenation QDa simultaneously quick detection health liquor, wine sample to be measured uses the Ultra Performance Liquid Chromatography instrument equipped with QDa detectors to be detected after pre-treatment, obtain the chromatogram of sample to be tested, qualitative analysis is carried out according to selection ion, quantitative analysis is then carried out to health liquor wine sample to be measured according to the standard curve of six kinds of glucides.The method of the present invention is simple and fast, accurately and reliably, available for detecting the content of 6 kinds of glucides (glucose, fructose, sucrose, lactose, maltose and inositol) in health liquor simultaneously, accurate judgement, quick detection for glucide in health liquor provide scientific basis.

Description

It is a kind of to utilize in ultra performance liquid chromatography concatenation QDa simultaneously quick detection health liquor six The method of kind glucide
Technical field
Six kinds of carbohydrate objects in ultra performance liquid chromatography concatenation QDa simultaneously quick detection health liquor are utilized the present invention relates to a kind of The method of matter belongs to detection and analysis technical field.
Background technology
Carbohydrate and glycitols are carbohydrate, are the natural components in food and health liquor, have important battalion Support value.In order to tart up or imitate fresh food, some carbohydrates can be added in the food after processing.In recent years, with hair Increasingly increase up to countries population's obesity and diabetes prevalence, the further monitoring demand of carbohydrate intake is also continuously increased. Therefore, it to meet increasingly harsh laws and regulations requirement, needs to provide accurate sugared content information on food labelling.According to existing document report The analysis of road, carbohydrate and glycitols is extremely challenging, because lacking chromophore in their compound structure, and various molecules Between quite similar, many of which isomers each other.
Existing technology is using liquid chromatograph (being furnished with RI or ELS detectors).RI testing requirements to mobile phase into Row Cautious control, to avoid any variation occurs in analysis, it is therefore desirable to isocratic elution.When being detected using RI, it is difficult to one Secondary analyze changes mobile phase composition between analysis next time, this is because when using different mobile phase compositions, RI detections Device may need a few hours to can be only achieved balance or even when using a collection of new identical mobile phase, and RI remains to detect tiny Variation and baseline is caused to change.In the case where mobile phase composition changes, ELS detections are in contrast relatively reliable, but It is the sensitivity and selectivity that ELS can not usually realize that carbohydrate detection is required in complicated food substrate, in view of the foregoing, establishes The method of a kind of while quick detection glucide is extremely urgent.The present invention concatenates QDa detectors using ultra performance liquid chromatography, The above-mentioned problem is completely avoided, greatly reduces the difficulty of carbohydrate analysis, is the accurate of glucide in health liquor wine Judgement, quick detection provide scientific basis.
Invention content
The present invention is the shortcoming present in order to avoid the above-mentioned prior art, it is desirable to provide a kind of to utilize ultra high efficiency liquid The method that phase chromatography concatenation QDa quickly detects six kinds of glucides in health liquor simultaneously.Six kinds of glucides for glucose, Fructose, sucrose, lactose, maltose and inositol.The method of the present invention can be the accurate judgement that glucide detects in health liquor, Quick detection provides scientific basis.
The present invention concatenates the side of six kinds of glucides in QDa while quick detection health liquor using ultra performance liquid chromatography Method includes the following steps:
Step 1:Pre-treatment
1.0~5.0mL health liquor wine samples to be measured are measured in 10mL plastic centrifuge tubes, adding in 8~10mL volumetric concentrations is 75% acetonitrile solution, be vortexed 1~5min, 1000 revs/min of 5~10min of centrifugation, 0.22 μm of filter membrane syringe filters filtering, Obtain sample to be tested;
Step 2:The drafting of standard curve
Prepare the 1000mg/L of five kinds of table sugars (fructose, glucose, sucrose, maltose and lactose) and inositol respectively with water Storing solution, then with acetonitrile solution (50:50, v/v) the mixing storing solution of dilution preparation 50mg/L (mixes each group in storing solution The concentration divided is 50mg/L);With acetonitrile solution (50:50, v/v) dilution gained mixes storing solution to obtain various concentration Hybrid standard working solution is detected using the Ultra Performance Liquid Chromatography instrument equipped with QDa detectors, with the peak area of determinand Linear regression is carried out to its corrresponding quality concentration, obtains the equation of linear regression of six kinds of glucides, each equation of linear regression institute Corresponding curve, the standard curve of as corresponding glucide;
In step 2, the point of a concentration of 5~1000ng/mL of hybrid standard working solution, at least 5 various concentrations of preparation Value.
In step 2, the testing conditions setting of Ultra Performance Liquid Chromatography instrument is as follows:
Chromatographic column:2.1 × 150mm, 1.7 μm of BEH Amide glucides analysis chromatographic column;Vortex instrument and liquid-transfering gun;
Column temperature is 30~50 DEG C;
Sample room temperature is 10~20 DEG C;
Mobile phase:A phases are 75% acetonitrile solution (A), and B phases are 10mmolNH4HCO3Solution (contains 0.1% NH4OH solution);
Flow velocity is 0.15~0.30mL/min;
Type of elution is isocratic elution, and mobile phase A is with Mobile phase B elution volume ratio:15:85;
Sample size is 0.5~2 μ L;
Detection time is 15~20min;
The setting condition of QDa detectors is:
System:ACQUITY QDa mass detectors, Performance patterns;
Ionization pattern:ESI-;
Capillary voltage:0.8V;
Orifice potential:4.0V;
Probe temperature:600℃;
Acquisition rate:1HZ;
Full scan:50-450m/z;
SIR mass numbers see the table below 1:
Table 1
Compound name SIR(m/z) Form the mode of ion
Fructose 215.1 [M+CL-]-
Glucose 215.1 [M+CL-]-
Inositol 179.2 [M-H+]-
Lactose 377.2 [M+CL-]-
Maltose 377.2 [M+CL-]-
Sucrose 341.3 [M-H+]-
* illustrate:Multiple mass number monitorings have been carried out to each carbohydrate.In order to improve sound of the substance in mass spectrum Should, reduce the detection limit and quantitative limit of substance, the present invention using novel formation ion mode, fructose, glucose, lactose and Maltose has used the mode of chlorination to form chlorine adduct.
Step 3:The detection of health liquor wine sample to be measured
1.0~2.0mL of sample to be tested that step 1 is taken to obtain, is detected using the identical testing conditions of step 2, is obtained The chromatogram of sample to be tested is obtained, qualitative analysis is carried out according to selection ion, then according to the standard curve of six kinds of glucides Quantitative analysis is carried out to health liquor wine sample to be measured.
Make following sensitivity test to the method for the present invention:The sensitivity of sensitivity test including instrument and method it is sensitive Degree, the detection limit of the sensitivity instrument of instrument represent, the Cmin of the mixed standard solution of signal-to-noise ratio >=3 is taken to be examined for instrument Rising limit;The quantitative limit of the sensitivity of method method represents that the Cmin for taking the mixed standard solution of signal-to-noise ratio >=9 is method Quantitative limit.The related data of gained is shown in Table 2.
Make following accuracy and reproducibility experiment to the method for the present invention:Same health liquor sample is selected to make after pre-treatment For blank sample, it is divided into 3 parts, is separately added into hybrid standard working solution and carries out recovery testu, calculate the rate of recovery;Choose 1 A health liquor sample handles 6 according to same pre-treating method, is tested respectively, by calculating its relative standard deviation (RSD) range carrys out the reproducibility of discriminatory analysis method.The accuracy of method is represented with the rate of recovery, is shown in Table 3, the reproduction of method Property is represented with relative standard deviation (RSD), is shown in Table 4.It can be seen that the rate of recovery is in 80~120%, RSD < 10%.
The recovery of standard addition experiment of 3 six kinds of glucides of table
The reproducibility experiment of 4 six kinds of sugar substances of table
The present invention is detected using QDa mass detectors, and using full scan pattern (SIR), sensitivity meets sample lower bound amount Testing requirements, while the detector is easy to use, plug and play does not need to be tuned, and can quickly detect in white wine Glucide and inositol.The detection limit and quantitative limit of six kinds of glucides are fully able to meet the low content of drinks sample, and measure will It asks, each compound rate of recovery, reproducibility are satisfied by quantitative test requirement.
Beneficial effects of the present invention are embodied in:
1st, the present invention establishes a kind of using in ultra performance liquid chromatography concatenation QDa detectors simultaneously quick detection health liquor The method of six kinds of glucides, can be accurately qualitative, quantitative to this six kinds of substances progress in white wine, is carbohydrate object in white wine Accurate judgement, the quick detection of matter provide scientific basis.
2nd, ultra performance liquid chromatography of the present invention concatenation QDa mass detectors it is simple and quick, accurately and reliably, repeatability It is good.
3rd, 2.1 × 150mm of the present invention, 1.7 μm of BEH Amide glucides analysis chromatographic column and acetonitrile and 10mmolNH4HCO3Solution (contains 0.1% NH4OH solution) selection of mobile phase reaches 6 kinds of glucides in health liquor Excellent separating effect.
4th, it by the present invention in that with QDa detectors, does not need to useC18 pillars are purified, direct injected Test, greatly reduces testing cost.
5th, pre-treatment operating procedure of the invention is few, and reproducibility and mark-on reclaims are preferable, so as to reduce experimental error simultaneously Improve the accuracy of data.
6th, the present invention improves the analysis selectivity of compound identification by the way that retention time and quality analysis are combined.
7th, the present invention can not only distinguish co-elute component by charge-mass ratio, but also can be on triangular web using more Kind method, and the rapid translating between different method conditions.
8th, this experiment input mode is direct injected, after analyzing chromatography post separation by BEH Amide glucides, directly It taps into mass detector, the sensitivity of method can be greatly improved, substantially reduce the detection limit and quantitative limit of method.
Description of the drawings
Fig. 1 is the liquid chromatogram of six kinds of glucide hybrid standard working solutions.
Specific embodiment
Technical solution of the present invention is further elaborated with reference to specific embodiment.
1st, pre-treatment
5.0mL health liquor wine samples to be measured are measured in 10mL plastic centrifuge tubes, add in the second that 10mL volumetric concentrations are 75% Nitrile solution, vortex 2min, 1000 revs/min centrifuge 5~10min, 0.22 μm of filter membrane syringe filters filtering, and test sample is treated in acquisition Product;
2nd, testing conditions are set
The testing conditions setting of Ultra Performance Liquid Chromatography instrument is as follows:
Chromatographic column:2.1 × 150mm, 1.7 μm of BEH Amide glucides analysis chromatographic column;Vortex instrument and liquid-transfering gun;
Column temperature is 40 DEG C;
Sample room temperature is 15 DEG C;
Mobile phase:A phases are 75% acetonitrile solution (A), and B phases are 10mmolNH4HCO3Solution (contains 0.1% NH4OH solution);
Flow velocity is 0.15mL/min;
Type of elution is isocratic elution, and mobile phase A is with Mobile phase B volume ratio during isocratic elution:15:85;
Sample size is 0.7 μ L;
Detection time is 17min;
The setting condition of QDa detectors is:
System:ACQUITY QDa mass detectors, Performance patterns;
Ionization pattern:ESI-;
Capillary voltage:0.8V;
Orifice potential:4.0V;
Probe temperature:600℃;
Acquisition rate:1HZ;
Full scan:50-450m/z;
SIR mass numbers see the table below 1:
Table 1
Compound name SIR(m/z) Form the mode of ion
Fructose 215.1 [M+CL-]-
Glucose 215.1 [M+CL-]-
Inositol 179.2 [M-H+]-
Lactose 377.2 [M+CL-]-
Maltose 377.2 [M+CL-]-
Sucrose 341.3 [M-H+]-
3rd, the drafting of standard curve
Prepare the 1000mg/L of five kinds of table sugars (fructose, glucose, sucrose, maltose and lactose) and inositol respectively with water Storing solution, then with acetonitrile solution (50:50, v/v) the mixing storing solution of dilution preparation 50mg/L (mixes each group in storing solution The concentration divided is 50mg/L);With acetonitrile solution (50:50, v/v) dilution gained mixes storing solution to obtain various concentration Hybrid standard working solution uses the Ultra Performance Liquid Chromatography instrument equipped with QDa detectors to be examined with the setup parameter of step 2 It surveys, linear regression is carried out to its corrresponding quality concentration with the peak area of determinand, obtains the linear regression side of six kinds of glucides Journey, the curve corresponding to each equation of linear regression, the standard curve of as corresponding glucide;
A concentration of 5~1000ng/mL of hybrid standard working solution, respectively 5ng/ml, 50ng/ml, 200ng/ml, 500ng/ml, 1000ng/ml;
The corresponding equation of linear regression of six kinds of glucides and correlation coefficient r2It see the table below 2.
The equation of linear regression of 2 six kinds of glucides of table, related coefficient, detection limit, quantitative limit
4th, the detection of health liquor wine sample to be measured
1.0~2.0mL of sample to be tested that step 1 is taken to obtain, is detected using the testing conditions of step 2, is obtained The chromatogram of sample to be tested carries out qualitative analysis according to selection ion, is then treated according to the standard curve of six kinds of glucides It surveys health liquor wine sample and carries out quantitative analysis.The detection data of this method see the table below 5 with existing method detection data correction data.
The detection data of 5 this method of table and existing method detection data correction data
It can be seen from the data in Table 5 that the same sample using existing method compared with this method, the detection limit of this method It is relatively low, it is able to detect that the content that existing method can't detect using this method, the content more than two methods detection limit makes It is able to detect that with both of which, the data of two methods detection, can be with by detection data in rational error range This method is absolutely proved better than existing method, accurately glucide in 6 can be quantified.

Claims (6)

1. a kind of method using six kinds of glucides in ultra performance liquid chromatography concatenation QDa simultaneously quick detection health liquor, It is characterized in that including the following steps:
Step 1:Pre-treatment
1.0~5.0mL health liquor wine samples to be measured are measured in 10mL plastic centrifuge tubes, it is 75% to add in 8~10mL volumetric concentrations Acetonitrile solution, be vortexed 1~5min, and 1000 revs/min of 5~10min of centrifugation, 0.22 μm of filter membrane syringe filters filtering obtains Sample to be tested;
Step 2:The drafting of standard curve
Prepare the 1000mg/L storing solutions of fructose, glucose, sucrose, maltose and lactose and inositol respectively with water, then with The mixing storing solution of 50mg/L is prepared in acetonitrile solution dilution;Gained mixing storing solution is diluted with acetonitrile solution to obtain difference The hybrid standard working solution of concentration, is detected using the Ultra Performance Liquid Chromatography instrument equipped with QDa detectors, with determinand Peak area carries out linear regression to its corrresponding quality concentration, obtains the equation of linear regression of six kinds of glucides, each linear regression Curve corresponding to equation, the standard curve of as corresponding glucide;
Step 3:The detection of health liquor wine sample to be measured
1.0~2.0mL of sample to be tested that step 1 is taken to obtain, is detected using the identical testing conditions of step 2, is obtained The chromatogram of sample to be tested carries out qualitative analysis according to selection ion, is then treated according to the standard curve of six kinds of glucides It surveys health liquor wine sample and carries out quantitative analysis.
2. according to the method described in claim 1, it is characterized in that:
In step 2, the volume ratio of acetonitrile and water is 50 in the acetonitrile solution:50.
3. according to the method described in claim 1, it is characterized in that:
In step 2, the point value of a concentration of 5~1000ng/mL of hybrid standard working solution, at least 5 various concentrations of preparation.
4. according to the method described in claim 1, it is characterized in that:
In step 2, the testing conditions setting of Ultra Performance Liquid Chromatography instrument is as follows:
Chromatographic column:2.1 × 150mm, 1.7 μm of BEH Amide glucides analysis chromatographic column;Vortex instrument and liquid-transfering gun;
Column temperature is 30~50 DEG C;
Sample room temperature is 10~20 DEG C;
Mobile phase:A phases are 75% acetonitrile solution, and B phases are 10mmolNH4HCO3Solution;
Flow velocity is 0.15~0.30mL/min;
Type of elution is isocratic elution;
Sample size is 0.5~2 μ L;
Detection time is 15~20min.
5. according to the method described in claim 4, it is characterized in that:
During isocratic elution, mobile phase A is with Mobile phase B elution volume ratio:15:85.
6. according to the method described in claim 1, it is characterized in that:
In step 2, the setting condition of QDa detectors is:
System:ACQUITY QDa mass detectors, Performance patterns;
Ionization pattern:ESI-;
Capillary voltage:0.8V;
Orifice potential:4.0V;
Probe temperature:600℃;
Acquisition rate:1HZ;
Full scan:50-450m/z.
CN201810201940.5A 2018-03-12 2018-03-12 Method for simultaneously and rapidly detecting six saccharides in health wine by connecting QDa in series through ultra-high performance liquid chromatography Active CN108169385B (en)

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Cited By (1)

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