CN105628825B - Based on UPC2The method of 4 kinds of principal monosaccharides and disaccharide content in ELSD measure honey - Google Patents
Based on UPC2The method of 4 kinds of principal monosaccharides and disaccharide content in ELSD measure honey Download PDFInfo
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Abstract
The invention discloses one kind to be based on UPC2The method of 4 kinds of principal monosaccharides and disaccharide content in ELSD measure honey；This method is that honey sample liquid is passed through into UPC successively2Separation and ELSD detections, quantitative analysis is carried out using external standard method, draw fructose in honey sample liquid, glucose, sucrose and the respective content of maltose, this method good separating effect, stability are strong, analysis time greatly shortens, cost is low, meet the requirement of green analysis detection, be advantageous to popularization and application.
Description
Technical field
The present invention relates to one kind to be based on UPC2- ELSD determines the method for 4 kinds of principal monosaccharides and disaccharide content in honey simultaneously,
More particularly to utilize UPC2The method that-ELSD determines Fructose in Honey, glucose, sucrose and maltose content simultaneously, belongs to food
Product detection technique field.
Background technology
Honey, it is a kind of sugared supersaturation material, its main component is 2 kinds of monose such as fructose, glucose, and submember is
2 kinds of disaccharide such as a small amount of sucrose, maltose.GB/T 14963-2011《National food safety standard honey》In require fructose and
Glucose two kinds of monose total content ＞ 60%, the general ＜ 5% of cane sugar content (remove " wood wasp honey, citrus honey, alfalfa honey, litchi
Cane sugar content outside branch honey, Osmanthus forrestii honey " allows to be less than 10%).China be the first in the world bee-keeping big country, bee colony quantity and
Main bee product outlet is ranked first in the world, and honey annual production surpasses 300,000 tons, and the health of development and the people for national economy is done
Positive contribution is gone out.Therefore, it is single, double to establish a kind of 4 kinds of content highest that is easy, fast and efficiently determining in honey
Sugar --- fructose, glucose, the method for sucrose and maltose, it will help honey product quality reaches above-mentioned standard, so as to drop
The hidden danger of low honey adulteration, strong technical support is provided to realize that honey big country is transformed into honey power.
Existing national standard GB/T 18932.22-2003《Fructose in Honey, glucose, sucrose, the measure side of maltose content
Method》It is middle that 4 kinds of sugared contents in honey are determined using liquid chromatogram-differential pulse polarograpll method, preferably divide though this method has
From degree and accuracy, but Composition distribution is extremely sensitive to temperature change so that the baseline stability time is longer, is also not suitable for gradient
Elution, and detection sensitivity is relatively low.
There is pertinent literature report at present, HPLC combination C-CAD or ELSD can be used, carry out isocratic elution to detect food
The method of middle saccharide compound, such as fructose, glucose, sucrose, maltose, lactose and maltotriose.But HPLC is used to tie
Close C-CAD progress isocratic elutions and substantial defect be present, such as time length, efficiency is low, and the sugared post or nh 2 column that HPLC is used are easy
Generation chemical degradation and cause test result stability relatively poor, so as to cause instrument loss big, cost is high, is unfavorable for promoting
Using.UPC2(Ultra-performance convergence chromatography, ultra high efficiency close phase chromatogram) is
The technical system based on supercritical fluid chromatography that Waters companies newly release, its mobile phase is with supercritical CO2Formed to be main,
With viscosity is low, molecule diffusion is fast, the advantage that separative efficiency is high, green.Chinese patent (Publication No. CN
105301131A) successfully by UPC2- ELSD is used to determine monoglyceride in molecular distillation glycerin monostearate, has easy, fast
The characteristics of fast, efficient.But at present not by UPC2- ELSD is used to detect any related report of saccharide compound in honey
Road, and saccharide compound is with maximum distinctive points existing for ester type compound, saccharide compound has more than ester type compound
Substantial amounts of isomer be present in monose or oligosaccharides in strong polarity, particularly carbohydrate etc., such as the fructose in monose and
Glucose, is isomer each other, sucrose and maltose in disaccharides, and isomer each other, therefore carbohydrate chemical combination
Thing is more difficult to realize separation than ester type compound.
The content of the invention
The defects of existing for the existing detection technique on saccharide compound in honey, the purpose of the present invention is to be
Good a kind of simple and effective, stability, Fast And Accurate Determination Fructose in Honey, glucose, sucrose and maltose method are provided.
In order to realize above-mentioned technical purpose, the invention provides one kind to be based on UPC2- ELSD determines 4 kinds of masters in honey simultaneously
The method for wanting monose and disaccharide content, this method are that honey sample solution is passed through into UPC successively2Separation and ELSD detections, are used
External standard method carries out quantitative analysis, draws fructose in honey sample, glucose, sucrose and the respective content of maltose.
Preferable scheme, comprises the following steps：(1) by the hybrid standard liquid of fructose, glucose, sucrose and maltose successively
By UPC2Separation and ELSD detections, standard curve is established according to testing result；(2) honey sample solution is passed through into UPC successively2
Separation and ELSD detection, establishing criteria curve to testing result carry out quantitative analysis, draw fructose in honey sample, glucose,
Sucrose and the respective content of maltose.
More preferably scheme, detect in hybrid standard liquid and honey sample solution processes, UPC2The conditional parameter of control is such as
Under：
Chromatographic column：ACQUITY UPC2BEH chromatographic columns, 100mm × 3.0mm, ethylene bridge particle, 1.7 μm；
Column temperature：50℃；
Sample room temperature：10℃；
Mobile phase A：Supercritical CO2；
Mobile phase B：Methanol solution, the NH containing percentage by volume 0.2%3·H2O；
Gradient：
Back pressure：1700psi；
Flow velocity：1.0mL/min；
Sampling volume：1μL；
The conditional parameter of ELSD detectors control is as follows：Carrier gas is nitrogen, and pressure 30psi, drift tube temperature is 50 DEG C.
More preferably scheme, described honey sample solution concentration are 0.0015~0.0020g/mL.
More preferably scheme, by fructose, glucose, sucrose and maltose after dissolved dilution, prepare a series of differences
The hybrid standard liquid of concentration.
Further preferred scheme, a series of hybrid standard liquid of various concentrations include：(1) fructose, glucose, sucrose and
The concentration of maltose is respectively 1000mg/L, 750mg/L, 300mg/L and 300mg/L hybrid standard liquid, (2) fructose, grape
Sugar, the concentration of sucrose and maltose are respectively 500mg/L, 375mg/L, 150mg/L and 150mg/L hybrid standard liquid, (3)
Fructose, glucose, the concentration of sucrose and maltose are respectively 250mg/L, 187.5mg/L, 75mg/L and 75mg/L mixing mark
Quasi- liquid, (4) fructose, glucose, the concentration of sucrose and maltose are respectively 125mg/L, 93.75mg/L, 37.5mg/L and 37.5
Mg/L hybrid standard liquid, (5) fructose, glucose, the concentration of sucrose and maltose be respectively 62.5mg/L, 46.875mg/L,
18.75mg/L and 18.75mg/L hybrid standard liquid.
Preferable scheme, the mass body of fructose in honey sample, glucose, sucrose and maltose is drawn by detection and analysis
Product concentration (unit, mg/L), then degree % is converted into by equation below：
In formula：
X is the degree of fructose, glucose, sucrose or maltose in honey sample, unit %；
W is the concentration of fructose, glucose, sucrose or maltose in external standard method honey sample, unit mg/L；
M be honey sample concentration, unit mg/L.
The present invention based on UPC2- ELSD determines the assay method of Fructose in Honey, glucose, sucrose and maltose simultaneously
Including step in detail below：
(1) sample preparation
0.015~0.02g honey sample is accurately weighed respectively in 10mL volumetric flasks, and it accurately weighs and is accurate to
0.0001g, first plus 0.5~1mL water is dissolved, then with volume ratio is 1:1 isopropanol:N-hexane constant volume, solution is through having
UPC is directly carried out after machine membrane filtration2Analysis.
(2) standard liquid is prepared
4 kinds of single, double sugared standard items storing solutions are prepared first, accurately weigh 50mg fructose, 75mg glucose, 15mg respectively
In sucrose, 15mg maltose (accurately weigh and be accurate to 0.1mg) to different 10mL volumetric flasks, first add 0.5~1mL water
Dissolved, then with volume ratio be 1:1 isopropanol:N-hexane constant volume, it is made into fructose, 7500mg/L that concentration is 5000mg/L
Glucose, 1500mg/L sucrose and the single standard product storing solution of maltose；
Secondly 4 kinds of sugared mixed standard solutions are prepared, pipette 2mL 5000mg/L fructose, the mg/L of 1mL 7500 respectively
Glucose, 2mL 1500mg/L sucrose, 2mL 1500mg/L maltose single standard items storing solution in 10mL volumetric flasks
In, it is 1 with volume ratio:1 isopropanol:N-hexane constant volume, it is 1000mg/L fructose, 750mg/L Portugal to be made into concentration successively
Grape sugar, 300mg/L sucrose, the mixed standard solution of 300mg/L maltose；
Series standard working solution is finally prepared again, is accurately pipetted 5mL mixed standard solutions in 10mL volumetric flasks, is used body
Product is than being 1:1 isopropanol:N-hexane constant volume, then dilute step by step, the series standard working solution for obtaining fructose is 62.5mg/L,
125mg/L、250mg/L、500mg/L、1000mg/L；The series standard working solution of glucose be 46.875mg/L,
93.75mg/L、187.5mg/L、375mg/L、750mg/L；The series standard working solution of sucrose and maltose is
18.75mg/L、37.5mg/L、75mg/L、150 mg/L、300mg/L。
(3)UPC2- ELSD while condition determination
Phase chromatogram-EISD is closed using ultra high efficiency to be measured standard working solution and sample solution,
Condition is as follows：
Chromatographic column：ACQUITY UPC2BEH chromatographic columns (100mm × 3.0mm, 1.7 μm)；
Column temperature：50℃；
Sample room temperature：10℃；
Mobile phase A：Supercritical CO2；
Mobile phase B：Methanol solution (the NH containing percentage by volume 0.2%3·H2O)；
Gradient：It is shown in Table 1；
Back pressure：1700psi；
Flow velocity：1.0mL/min；
Sampling volume：1μL；
ELSD detectors：Carrier gas is nitrogen, and pressure 30psi, drift tube temperature is 50 DEG C.
Table 1
(4) calculating of measurement result
Utilize UPC2Separated, ELSD is measured, quantified by external standard method, with peak area (Y) to mass concentration (mg/L)
Linear regression is carried out, equation of linear regression is obtained, the honey sample prepared is measured, by the softwares of Empower 3.0,
The automatic fructose respectively obtained in sample, glucose, the content (mg/L) of sucrose and maltose, then it is converted into degree
(%), its calculation formula are：
In formula：
X is the degree of fructose, glucose, sucrose or maltose in honey sample, unit %；
W is the concentration of fructose, glucose, sucrose or maltose in external standard method honey sample, unit mg/L；
M be honey sample concentration, unit mg/L.
Compared with the prior art, the beneficial effect that technical scheme is brought：
(1) present invention utilizes UPC first2- ELSD technologies determine 4 kinds of main single, double sugared contents, UPC in honey simultaneously2-
ELSD greatly shortens to the gradient elution good separating effects of various carbohydrates, analysis time, and test result is stable, high sensitivity,
Realize the fast and accurately measure of 4 kinds of carbohydrates in honey；
(2) technical scheme is adapted to separating polar using the BEH chromatographic columns of sub- 2 μm ethylene bridge particle
Strong glucide, good separation, measurement result is stable, in the absence of the other sugared post such as nh 2 columns of puzzlement, efficiently sugared post etc.
The chemical degradation problem of appearance；Use supercritical CO simultaneously2Make main mobile phase, operating cost is low, more conforms to green
Analyze the requirement of detection.
Brief description of the drawings
【Fig. 1】For concentration in the embodiment of the present invention 1 be respectively 1000mg/L fructose, 750mg/L glucose,
The chromatogram of 300mg/L sucrose and the mixed standard solution of 300mg/L maltose.
【Fig. 2】For the chromatogram of honey sample A in the embodiment of the present invention 1.
【Fig. 3】For the chromatogram of honey sample B in the embodiment of the present invention 2.
Embodiment
Present invention is described in detail below in conjunction with accompanying drawing embodiment, it is intended to helps reader to more fully understand the present invention
Essence, but implementation that can not be to the present invention and protection domain form any restriction.
Embodiment 1：
(1) chromatographic condition
Chromatographic column is ACQUITY UPC2BEH (100mm × 3.0mm, 1.7 μm)；System back pressure is 1700 psi；Chromatographic column
Temperature is 50 DEG C；Sample room temperature：10℃；Mobile phase is supercritical CO2(mobile phase A) and methanol solution (contain percentage by volume
0.2% NH3·H2O) (Mobile phase B), gradient table 1 for example listed hereinbefore；Flow velocity is 1.0mL/min；Sampling volume：1μL.
Using ELS as detector, carrier gas is nitrogen, and pressure 30psi, drift tube temperature is 50 DEG C, gain 500.
(2) standard liquid is prepared
Accurately weigh 50mg fructose, 75mg glucose, 15mg sucrose, 15mg maltose respectively first and (accurately weigh essence
Really to 0.1mg) in different 10mL volumetric flasks, first plus 0.5~1mL water is dissolved, then with volume ratio is 1:1 isopropyl
Alcohol:N-hexane constant volume, concentration is made into as 5000mg/L fructose, 7500mg/L glucose, 1500mg/L sucrose and malt
The single standard product storing solution of sugar；Secondly 2mL 5000mg/L fructose, 1mL 7500mg/L glucose, 2mL are pipetted respectively
1500mg/L sucrose, single standard items storing solution of 2mL 1500mg/L maltose constant volume, are matched somebody with somebody successively in 10mL volumetric flasks
Into the hybrid standard that concentration is 1000mg/L fructose, 750mg/L glucose, 300mg/L sucrose, 300mg/L maltose
Solution；Finally using volume ratio as 1:1 isopropanol:N-hexane is solvent, using mixed standard solution dilution method, is made into successively
1000/750/300/300mg/L、500/375/150/150mg/L、 250/187.5/75/75mg/L、125/93.75/37.5/
37.5mg/L, 62.5/46.875/18.75/18.75mg/L standard liquid, mass concentration is carried out with standard series peak area
Linear regression, equation of linear regression is obtained, as shown in table 2, standard liquid chromatogram is as shown in Figure 1.
24 kinds of table single, double sugared equation of linear regression and detection limit
Y：Peak area；X：Mass concentration：mg/L；Detection limit：Signal to noise ratio S/N=3
(3) sample preparation
Commercially available 15~20mg of honey sample (being accurate to 0.0001g) is accurately weighed respectively in 10mL volumetric flasks, is first added
0.5~1mL water is dissolved, then with volume ratio be 1:1 isopropanol:N-hexane constant volume, solution are straight after organic membrane filter
Tap into row UPC2Analysis.
(4) sample analysis
Using this method to 4 kinds of single, double sugar --- fructose, glucose, sucrose and the malt in commercially available honey (sample A)
Sugar is measured, and measurement result is as shown in table 3, and the fructose and glucose total content in sample are more than 60%, and cane sugar content is less than
5%, meet honey national standard, sample chromatogram figure is as shown in Figure 2；
The sample A testing results of table 3
| Key component | Content (%) |
| Fructose | 41.60 |
| Glucose | 24.75 |
| Sucrose | 2.26 |
| Maltose | 0.81 |
Embodiment 2：
Specific steps as described in Example 1, select another commercially available honey (sample B) to be measured, and measure its 4 kinds mainly
Single, double sugared content be shown in Table 4, the fructose and glucose total content in sample are 64.70%, and cane sugar content is less than 5%, meets honeybee
Sweet national standard, sample chromatogram figure as shown in Figure 3
The sample B testing results of table 4
| Key component | Content (%) |
| Fructose | 30.65 |
| Glucose | 34.05 |
| Sucrose | 3.28 |
| Maltose | 4.15 |
Embodiment 3：
The standard liquid recovery of standard addition that high, medium and low 3 concentration levels are carried out into the honey sample of known content is real
Test.Each mark-on horizontal parallel determines 5 times, and testing result is as shown in table 5, the rate of recovery of method 86.56%~
103.77%, RSD can meet the needs of actual sample measure 1.75%~4.29%.
4 kinds of single, double sugared recovery of standard addition and relative standard deviation (n=6) in the sample of table 5
Embodiment described above is only that the preferred embodiment of the present invention is described, not to the model of the present invention
Enclose and be defined, on the premise of design spirit of the present invention is not departed from, technical side of the those of ordinary skill in the art to the present invention
The various modifications and improvement that case is made, it all should fall into the protection domain of claims of the present invention determination.
Claims (5)
1. it is based on UPC2- ELSD determines the method for 4 kinds of principal monosaccharides and disaccharide content in honey simultaneously, it is characterised in that：Including with
Lower step：
(1) hybrid standard liquid of fructose, glucose, sucrose and maltose is passed through into UPC successively2Separation and ELSD detections, according to inspection
Survey result and establish standard curve；
(2) honey sample solution is passed through into UPC successively2Separation and ELSD detections, establishing criteria curve quantify to testing result
Analysis, draws fructose in honey sample, glucose, sucrose and the respective content of maltose；Described UPC2The condition ginseng of control
Number is as follows：
Chromatographic column：ACQUITY UPC2BEH chromatographic columns, 100mm × 3.0mm, ethylene bridge particle, 1.7 μm；
Column temperature：50℃；
Sample room temperature：10℃；
Mobile phase A：Supercritical CO2；
Mobile phase B：Methanol solution, the NH containing percentage by volume 0.2%3·H2O；
Gradient：
Back pressure：1700psi；
Flow velocity：1.0mL/min；
Sampling volume：1μL；
The conditional parameter of ELSD detectors control is as follows：Carrier gas is nitrogen, and pressure 30psi, drift tube temperature is 50 DEG C.
2. according to claim 1 be based on UPC2- ELSD determines the side of 4 kinds of principal monosaccharides and disaccharide content in honey simultaneously
Method, it is characterised in that：Described honey sample solution concentration is 0.0015~0.0020g/mL.
3. according to claim 1 be based on UPC2- ELSD determines the side of 4 kinds of principal monosaccharides and disaccharide content in honey simultaneously
Method, it is characterised in that：By fructose, glucose, sucrose and maltose after dissolved dilution, a series of the mixed of various concentrations is prepared
Standardization liquid.
4. according to claim 3 be based on UPC2- ELSD determines the side of 4 kinds of principal monosaccharides and disaccharide content in honey simultaneously
Method, it is characterised in that：A series of hybrid standard liquid of various concentrations includes：(1) fructose, glucose, sucrose and maltose is dense
Degree respectively 1000mg/L, 750mg/L, 300mg/L and 300mg/L hybrid standard liquid, (2) fructose, glucose, sucrose and wheat
Bud sugar concentration be respectively 500mg/L, 375mg/L, 150mg/L and 150mg/L hybrid standard liquid, (3) fructose, glucose,
The concentration of sucrose and maltose is respectively 250mg/L, 187.5mg/L, 75mg/L and 75mg/L hybrid standard liquid, (4) fructose,
The concentration of glucose, sucrose and maltose is respectively 125mg/L, 93.75mg/L, 37.5mg/L and 37.5mg/L hybrid standard
Liquid, (5) fructose, glucose, the concentration of sucrose and maltose be respectively 62.5mg/L, 46.875mg/L, 18.75mg/L and
18.75mg/L hybrid standard liquid.
5. according to any one of Claims 1 to 4 based on UPC2- ELSD determines 4 kinds of principal monosaccharides and disaccharide in honey simultaneously
The method of content, it is characterised in that：Fructose in honey sample, glucose, sucrose and maltose are drawn by detection and analysis each
Mass-volume concentration, then degree % is converted into by equation below：
In formula：
X is the degree of fructose, glucose, sucrose or maltose in honey sample, unit %；
W is the concentration of fructose, glucose, sucrose or maltose in external standard method honey sample, unit mg/L；
M be honey sample concentration, unit mg/L.
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