CN105628825A - Method for measuring contents of four main monosaccharides and disaccharides in honey on basis of UPC2-ELSD - Google Patents

Abstract

The invention discloses a method for measuring the contents of four main monosaccharides and disaccharides in honey on the basis of UPC2-ELSD. According to the method, honey sample liquid is sequentially subjected to UPC2 separation and ELSD detection, quantitative analysis is conducted through an external standard method, and the contents of fructose, glucose, cane sugar and maltose in the honey sample liquid are obtained. The method is good in separation effect, high in stability and low in cost, analysis time is greatly shortened, the requirement for environment-friendly analysis and detection is met, and application and popularization are facilitated.

Description

Based on UPC2-ELSD measures the method for 4 kinds of principal monosaccharides and disaccharide content in honey

Technical field

The present invention relates to a kind of based on UPC²The method of 4 kinds of principal monosaccharides and disaccharide content in-ELSD Simultaneously test honey, in particular to utilizing UPC²In-ELSD Simultaneously test honey, the method for fructose, glucose, sucrose and maltose content, belongs to technical field of food detection.

Background technology

Honey is the supersaturation material of a kind of sugar, and 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/T14963-2011 " national food safety standard honey " requires fructose and these two kinds of monose total content > 60% of glucose, the general < 5% (sucrose content except " wood wasp honey, oranges and tangerines honey, alfalfa honey, lichee honey, Osmanthus forrestii honey " allows to be less than 10%) of sucrose content. China be the first in the world bee-keeping big country, bee colony quantity and main bee product outlet all rank first in the world, honey annual production surpasses 300,000 tons, for the development of national economy and the health of the people have made positive contribution. Therefore, set up a kind of simplicity, measure the method for 4 kinds of the highest single, double candy sugar of the content in honey, glucose, sucrose and maltose fast and efficiently, honey product quality will be contributed to reach above-mentioned standard, thus reduce the hidden danger of honey adulteration, for realizing the technical support that honey big country is transformed into honey power and provides strong.

Existing GB GB/T18932.22-2003 " in honey the measuring method of fructose, glucose, sucrose, maltose content " adopt liquid chromatography-differential pulse polarograpll method measure these 4 kinds of sugared content in honey, though the method has good resolution and accuracy, but Composition distribution to temperature variation extremely sensitivity make the baseline stability time longer, also gradient elution is not suitable for, and detection sensitivity is lower.

Pertinent literature is had to report at present, it is possible to adopt HPLC in conjunction with C-CAD or ELSD, carry out isocratic elution to detect the method for saccharide compound in food, such as fructose, glucose, sucrose, maltose, lactose and trisaccharide maltose etc. But adopting HPLC to carry out isocratic elution in conjunction with C-CAD and there is substantive defect, as the time is long, efficiency is low, easily there is chemical degradation and cause test result stability relatively poor in the sugared post that HPLC uses or nh 2 column, thus causing instrument loss big, cost height, is unfavorable for applying. UPC²(Ultra-performanceconvergencechromatography, ultra-high efficiency closes phase chromatogram) is the technical system based on supercritical fluid chromatography that Waters company newly releases, and its moving phase is with supercritical CO₂For main composition, have that viscosity is low, molecular diffusion is fast, separation efficiency height, environmental protection advantage. Chinese patent (publication number is CN105301131A) is successfully by UPC²ELSD, for measuring mono-glycerides in molecular distillation glyceryl monostearate, has simplicity, fast and efficiently feature. But at present not by UPC²ELSD is for detecting any relevant report of saccharide compound in honey, and the maximum distinctive points that saccharide compound and ester compound exist is, saccharide compound has stronger polarity than ester compound, particularly there is a large amount of isomerss in the monose in carbohydrate or oligosaccharides etc., such as the fructose in monose and glucose, it is isomers each other, sucrose in disaccharides and maltose, also it is isomers, therefore saccharide compound realization separation more difficult than ester compound each other.

Summary of the invention

For the defect that the existing detection technique about saccharide compound in honey exists, it is an object of the invention to be to provide a kind of fructose, glucose, sucrose and maltose method in simple and effective, good stability, fast accurate mensuration honey.

In order to realize above-mentioned technical purpose, the present invention provides a kind of based on UPC²The method of 4 kinds of principal monosaccharides and disaccharide content in-ELSD Simultaneously test honey, the method is successively through UPC by honey sample solution²Separation and ELSD detection, adopt external standard method to carry out quantitative analysis, draw fructose in honey sample, glucose, sucrose and maltose content separately.

Preferred scheme, comprises the following steps: (1) by the hybrid standard liquid of fructose, glucose, sucrose and maltose successively through UPC²Separation and ELSD detection, according to detected result Criterion curve; (2) by honey sample solution successively through UPC²Separation and ELSD detection, detected result is carried out quantitative analysis by establishing criteria curve, draws fructose in honey sample, glucose, sucrose and maltose content separately.

In more preferably scheme, detection hybrid standard liquid and honey sample solution processes, UPC²The conditional parameter of control is as follows:

Chromatographic column: ACQUITYUPC²BEH chromatographic column, 100mm �� 3.0mm, ethylene bridge particle, 1.7 ��m;

Post temperature: 50 DEG C;

Sample chamber temperature: 10 DEG C;

Mobile phase A: supercritical CO₂;

Mobile phase B: methanol solution, containing the NH of percent by volume 0.2%₃��H₂O;

Gradient:

Back pressure: 1700psi;

Flow velocity: 1.0mL/min;

Sampling volume: 1 �� L;

The conditional parameter of ELSD detector control is as follows: carrier gas is nitrogen, and pressure is 30psi, and drift tube temperature is 50 DEG C.

More preferably scheme, described honey sample strength of solution is 0.0015��0.0020g/mL.

More preferably scheme, by fructose, glucose, sucrose and maltose after dissolved dilution, prepares the hybrid standard liquid of a series of different concns.

Preferred scheme further, the hybrid standard liquid of a series of different concns comprises: (1) fructose, glucose, the concentration of sucrose and maltose is respectively 1000mg/L, 750mg/L, the hybrid standard liquid of 300mg/L and 300mg/L, (2) fructose, glucose, the concentration of sucrose and maltose is respectively 500mg/L, 375mg/L, the hybrid standard liquid of 150mg/L and 150mg/L, (3) fructose, glucose, the concentration of sucrose and maltose is respectively 250mg/L, 187.5mg/L, the hybrid standard liquid of 75mg/L and 75mg/L, (4) fructose, glucose, the concentration of sucrose and maltose is respectively 125mg/L, 93.75mg/L, the hybrid standard liquid of 37.5mg/L and 37.5mg/L, (5) fructose, glucose, the concentration of sucrose and maltose is respectively 62.5mg/L, 46.875mg/L, the hybrid standard liquid of 18.75mg/L and 18.75mg/L.

Preferred scheme, analyzed the quality volumetric concentration (unit, mg/L) drawing fructose in honey sample, glucose, sucrose and maltose by detection, then it is converted into degree % by following formula:

$X\%=\frac{w}{m}\&times;100$

In formula:

X is the degree of fructose, glucose, sucrose or maltose in honey sample, and unit is %;

W is the concentration of fructose, glucose, sucrose or maltose in external standard method honey sample, and unit is mg/L;

M is the concentration of honey sample, and unit is mg/L.

The present invention based on UPC²In-ELSD Simultaneously test honey, the measuring method of fructose, glucose, sucrose and maltose comprises following concrete steps:

(1) sample preparation

The honey sample accurately taking 0.015��0.02g respectively is in 10mL volumetric flask, it accurately takes and is accurate to 0.0001g, first adding 0.5��water of 1mL dissolved, then is the Virahol of 1:1 by volume ratio: normal hexane holds surely, and solution directly carries out UPC after organic membrane filter²Analyze.

(2) standardized solution preparation

First the standard substance storing solution of 4 kinds of single, double sugar is prepared, accurately take 50mg fructose, 75mg glucose, 15mg sucrose, 15mg maltose (all accurately take and be accurate to 0.1mg) respectively in different 10mL volumetric flasks, first adding 0.5��water of 1mL dissolved, it is the Virahol of 1:1 by volume ratio again: normal hexane holds surely, is made into the single standard product storing solution of fructose, the glucose of 7500mg/L, the sucrose of 1500mg/L and maltose that concentration is 5000mg/L;

Secondly preparation 4 kinds of sugared mixed standard solutions, pipette respectively the fructose of 2mL5000mg/L, the glucose of 1mL7500mg/L, the sucrose of 2mL1500mg/L, the maltose of 2mL1500mg/L single standard substance storing solution in 10mL volumetric flask, it is the Virahol of 1:1 by volume ratio: normal hexane holds surely, it is made into fructose that concentration is 1000mg/L, the glucose of 750mg/L, the sucrose of 300mg/L, the mixed standard solution of 300mg/L maltose successively;

Finally prepare series standard working solution again, accurately pipette 5mL mixed standard solution in 10mL volumetric flask, it is the Virahol of 1:1 by volume ratio: normal hexane holds surely, stepwise dilution again, the series standard working solution obtaining fructose is 62.5mg/L, 125mg/L, 250mg/L, 500mg/L, 1000mg/L; The series standard working solution of glucose is 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, 150mg/L, 300mg/L.

(3)UPC²-ELSD Simultaneously test condition

Utilizing ultra-high efficiency to close phase chromatogram-light scattering detector standard operation solution and sample solution to be measured, condition is as follows:

Chromatographic column: ACQUITYUPC²BEH chromatographic column (100mm �� 3.0mm, 1.7 ��m);

Post temperature: 50 DEG C;

Sample chamber temperature: 10 DEG C;

Mobile phase A: supercritical CO₂;

Mobile phase B: methanol solution is (containing the NH of percent by volume 0.2%₃��H₂O);

Gradient: in table 1;

Back pressure: 1700psi;

Flow velocity: 1.0mL/min;

Sampling volume: 1 �� L;

ELSD detector: carrier gas is nitrogen, pressure is 30psi, and drift tube temperature is 50 DEG C.

Table 1

(4) calculating of measurement result

Utilize UPC²It is separated, ELSD measures, quantified by external standard method, with peak area (Y), mass concentration (mg/L) is carried out linear regression, obtain equation of linear regression, the honey sample prepared is measured, by Empower3.0 software, automatically obtaining the content (mg/L) of the fructose in sample, glucose, sucrose and maltose respectively, then be converted into degree (%), its calculation formula is:

$X\%=\frac{w}{m}\&times;100$

In formula:

X is the degree of fructose, glucose, sucrose or maltose in honey sample, and unit is %;

W is the concentration of fructose, glucose, sucrose or maltose in external standard method honey sample, and unit is mg/L;

M is the concentration of honey sample, and unit is mg/L.

Hinge structure, the useful effect that the technical scheme of the present invention is brought:

(1) the present invention utilizes UPC first²4 kinds of main single, double sugar content in-ELSD technology Simultaneously test honey, UPC²Gradient elution separation effective to various carbohydrate of-ELSD, analysis time shortens greatly, and test result is stablized, highly sensitive, it is achieved that the mensuration fast and accurately of 4 kinds of carbohydrates in honey;

(2) technical scheme of the present invention adopts the BEH chromatographic column of the ethylene bridge particle of sub-2 ��m, it is applicable to the glucide that separating polar is strong, good separation, measurement result is stablized, and there is not the chemical degradation problem that other sugar post of puzzlement occurs such as nh 2 column, efficient sugar post etc.; Adopt supercritical CO simultaneously₂Making main moving phase, running cost is low, more meets the requirement that detection is analyzed in environmental protection.

Accompanying drawing explanation

[Fig. 1] is respectively the color atlas of the mixed standard solution of the fructose of 1000mg/L, the glucose of 750mg/L, the sucrose of 300mg/L and 300mg/L maltose for concentration in the embodiment of the present invention 1.

[Fig. 2] is the color atlas of honey sample A in the embodiment of the present invention 1.

[Fig. 3] is the color atlas of honey sample B in the embodiment of the present invention 2.

Embodiment

Below in conjunction with accompanying drawing embodiment, content of the present invention is described in detail, it is intended to help reader to understand the essence of the present invention better, but the enforcement of the present invention and protection domain can not be formed any restriction.

Embodiment 1:

(1) chromatographic condition

Chromatographic column is ACQUITYUPC²BEH (100mm �� 3.0mm, 1.7 ��m); System back pressure is 1700psi; Chromatogram column temperature is 50 DEG C; Sample chamber temperature: 10 DEG C; Moving phase is supercritical CO₂(mobile phase A) and methanol solution are (containing the NH of percent by volume 0.2%₃��H₂O) (Mobile phase B), gradient is such as table 1 listed hereinbefore; Flow velocity is 1.0mL/min; Sampling volume: 1 �� L. Adopting ELS as detector, carrier gas is nitrogen, and pressure is 30psi, and drift tube temperature is 50 DEG C, and gain is 500.

(2) standardized solution preparation

First 50mg fructose, 75mg glucose, 15mg sucrose, 15mg maltose (all accurately take and be accurate to 0.1mg) is accurately taken respectively in different 10mL volumetric flasks, first adding 0.5��water of 1mL dissolved, it is the Virahol of 1:1 by volume ratio again: normal hexane holds surely, is made into the single standard product storing solution of fructose, the glucose of 7500mg/L, the sucrose of 1500mg/L and maltose that concentration is 5000mg/L; Secondly pipette respectively the fructose of 2mL5000mg/L, the glucose of 1mL7500mg/L, the sucrose of 2mL1500mg/L, the maltose of 2mL1500mg/L single standard substance storing solution in 10mL volumetric flask, fixed hold, it is made into fructose that concentration is 1000mg/L, the glucose of 750mg/L, the sucrose of 300mg/L, the mixed standard solution of 300mg/L maltose successively; It is finally the Virahol of 1:1 taking volume ratio: normal hexane is solvent, adopt mixed standard solution dilution method, it is made into the standardized solution of 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 successively, with standard series peak area, mass concentration is carried out linear regression, obtain equation of linear regression, as shown in table 2, standardized solution color atlas is as shown in Figure 1.

The equation of linear regression of the single, double sugar of table 24 kind and detection limit

Y: peak area; X: mass concentration: mg/L; Detection limit: signal to noise ratio S/N=3

(3) sample preparation

Accurately take commercially available honey sample 15��20mg (being accurate to 0.0001g) respectively in 10mL volumetric flask, first adding 0.5��water of 1mL dissolved, it is the Virahol of 1:1 by volume ratio again: normal hexane holds surely, and solution directly carries out UPC after organic membrane filter²Analyze.

(4) sample analysis

Present method is adopted 4 kinds of single, double candy sugar in commercially available honey (sample A), glucose, sucrose and maltose to be measured, measurement result is as shown in table 3, fructose and glucose total content in sample are greater than 60%, sucrose content is less than 5%, meeting honey national standard, sample chromatogram figure is as shown in Figure 2;

Table 3 sample A detected result

Main ingredient	Content (%)
		Fructose	41.60
Glucose	24.75
		Sucrose	2.26
Maltose	0.81

Embodiment 2:

Concrete steps are as described in Example 1, the honey (sample B) selecting another commercially available measures, record its 4 kinds main single, double sugar content in table 4, fructose and glucose total content in sample are 64.70%, sucrose content is less than 5%, meeting honey national standard, sample chromatogram figure is as shown in Figure 3.

Table 4 sample B detected result

Main ingredient	Content (%)
		Fructose	30.65
Glucose	34.05
		Sucrose	3.28
Maltose	4.15

Embodiment 3:

The standardized solution recovery of standard addition experiment of high, medium and low 3 concentration levels is carried out in the honey sample of known content. Each adds mark horizontal parallel and measures 5 times, and detected result is as shown in table 5, and the rate of recovery of method 1.75%��4.29%, can meet the needs that actual sample measures at 86.56%��103.77%, RSD.

The recovery of standard addition of 4 kinds of single, double sugar and relative standard deviation (n=6) in table 5 sample

Above-described embodiment is only the preferred embodiment of the present invention be described; not the scope of the present invention is limited; under the prerequisite not departing from inventive design spirit; the various distortion that the technical scheme of the present invention is made by those of ordinary skill in the art and improvement, all should fall in the protection domain that claims of the present invention is determined.

Claims (7)

1. based on UPC²The method of 4 kinds of principal monosaccharides and disaccharide content in-ELSD Simultaneously test honey, it is characterised in that: honey sample solution is successively through UPC²Separation and ELSD detection, adopt external standard method to carry out quantitative analysis, draw fructose in honey sample, glucose, sucrose and maltose content separately.

2. according to claim 1 based on UPC²The method of 4 kinds of principal monosaccharides and disaccharide content in-ELSD Simultaneously test honey, it is characterised in that: comprise the following steps: (1) by the hybrid standard liquid of fructose, glucose, sucrose and maltose successively through UPC²Separation and ELSD detection, according to detected result Criterion curve; (2) by honey sample solution successively through UPC²Separation and ELSD detection, detected result is carried out quantitative analysis by establishing criteria curve, draws fructose in honey sample, glucose, sucrose and maltose content separately.

3. according to claim 2 based on UPC²The method of 4 kinds of principal monosaccharides and disaccharide content in-ELSD Simultaneously test honey, it is characterised in that: described UPC²The conditional parameter of control is as follows:

Chromatographic column: ACQUITYUPC²BEH chromatographic column, 100mm �� 3.0mm, ethylene bridge particle, 1.7 ��m;

Post temperature: 50 DEG C;

Sample chamber temperature: 10 DEG C;

Mobile phase A: supercritical CO₂;

Mobile phase B: methanol solution, containing the NH of percent by volume 0.2%₃��H₂O;

Gradient:

Back pressure: 1700psi;

Flow velocity: 1.0mL/min;

Sampling volume: 1 �� L;

The conditional parameter of ELSD detector control is as follows: carrier gas is nitrogen, and pressure is 30psi, and drift tube temperature is 50 DEG C.

4. according to claim 2 based on UPC²The method of 4 kinds of principal monosaccharides and disaccharide content in-ELSD Simultaneously test honey, it is characterised in that: described honey sample strength of solution is 0.0015��0.0020g/mL.

5. according to claim 2 based on UPC²The method of 4 kinds of principal monosaccharides and disaccharide content in-ELSD Simultaneously test honey, it is characterised in that: by fructose, glucose, sucrose and maltose after dissolved dilution, prepare the hybrid standard liquid of a series of different concns.

6. according to claim 5 based on UPC²The method of 4 kinds of principal monosaccharides and disaccharide content in-ELSD Simultaneously test honey, it is characterized in that: the hybrid standard liquid of a series of different concns comprises: (1) fructose, glucose, the concentration of sucrose and maltose is respectively 1000mg/L, 750mg/L, the hybrid standard liquid of 300mg/L and 300mg/L, (2) fructose, glucose, the concentration of sucrose and maltose is respectively 500mg/L, 375mg/L, the hybrid standard liquid of 150mg/L and 150mg/L, (3) fructose, glucose, the concentration of sucrose and maltose is respectively 250mg/L, 187.5mg/L, the hybrid standard liquid of 75mg/L and 75mg/L, (4) fructose, glucose, the concentration of sucrose and maltose is respectively 125mg/L, 93.75mg/L, the hybrid standard liquid of 37.5mg/L and 37.5mg/L, (5) fructose, glucose, the concentration of sucrose and maltose is respectively 62.5mg/L, 46.875mg/L, the hybrid standard liquid of 18.75mg/L and 18.75mg/L.

7. according to the arbitrary item of claim 1��6 based on UPC²Monose in-ELSD Simultaneously test honey and the method for disaccharide content, it is characterised in that: analyzed the quality volumetric concentration drawing fructose in honey sample, glucose, sucrose and maltose separately by detection, then it is converted into degree % by following formula:

$X\%=\frac{w}{m}\&times;100$

In formula:

X is the degree of fructose, glucose, sucrose or maltose in honey sample, and unit is %;

W is the concentration of fructose, glucose, sucrose or maltose in external standard method honey sample, and unit is mg/L;

M is the concentration of honey sample, and unit is mg/L.