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This standard replaces GB/T 5009.8-2008 Determination of Saccharose in Foods, GB/T 18932.22-2003 Method for the Determination of Fructose, Glucose, Sucrose, Maltose Contents in Honey - Liquid Chromatography Refractive Index Detection Method, and GB/T 22221-2008 Determination of Fructose, Glucose, Sucrose, Maltose, Lactose in Foods - High-Performance Liquid Chromatography.
The following main changes have been made with respect to GB/T 5009.8-2008:
——the standard name is revised as "食品安全国家标准 食品中果糖、葡萄糖、蔗糖、麦芽糖、乳糖的测定 (National Food Safety Standard Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Foods)";
——the pretreatment of partial samples is added.
National Food Safety Standard
Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Foods
1 Scope
This standard specifies the determination methods of fructose, glucose, sucrose, maltose and lactose in foods.
In this standard, Method I is applicable to the determination of fructose, glucose, sucrose, maltose and lactose in foods, and Method II is applicable to the determination of sucrose in foods.
"Method I" - high performance liquid chromatography is applicable to the determination of fructose, glucose, sucrose, maltose and lactose in such foods as cereals, milk products, fruit and vegetable products, bee honey, syrup and beverage.
"Method II" - acid hydrolysis - Lane-Eynon's method is applicable to the determination of sucrose in foods.
Method I High Performance Liquid Chromatography
2 Theory
After extraction, the fructose, glucose, sucrose, maltose and lactose in the specimen are separated with the high performance liquid chromatography column, and detected with refractive index detector or evaporative light-scattering detector, and subjected to quantitation by external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically pure reagents and Grade 1 water (specified in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Acetonitrile: chromatographically pure.
3.1.2 Zinc acetate [Zn(CH3COO)2·2H2O].
3.1.3 Potassium ferrocyanide {K4[Fe(CN)6]·3H2O}.
3.1.4 Petroleum ether: boiling range of 30℃~60℃.
3.2 Preparation of reagents
3.2.1 Zinc acetate solution: weigh 21.9g of zinc acetate, add 3mL of glacial acetic acid, dissolve them with water and dilute the solution to 100mL.
3.2.2 Potassium ferricyanide solution: weigh 10.6g of potassium ferricyanide, dissolve it with water and dilute the solution to 100mL.
3.3 Standards
3.3.1 Fructose (C6H12O6, CAS No.: 57-48-7) with the purity of 99%, or reference material approved and awarded with reference material certificate by the nation.
3.3.2 Glucose (C6H12O6, CAS No.: 50-99-7) with the purity of 99%, or reference material approved and awarded with reference material certificate by the nation.
3.3.3 Sucrose (C12H22O11, CAS No.: 57-50-1) with the purity of 99%, or reference material approved and awarded with reference material certificate by the nation.
3.3.4 Maltose (C12H22O11, CAS No.: 69-79-4) with the purity of 99%, or reference material approved and awarded with reference material certificate by the nation.
3.3.5 Lactose (C6H12O6, CAS No.: 63-42-3) with the purity of 99%, or reference material approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solutions
3.4.1 Standard stock solution of sugar (20mg/mL): separately weigh 1g of the above-mentioned fructose, glucose, sucrose, maltose and lactose which have been dried for 2h at 96℃±2℃, add water and scale the volume to 50mL, put in a place at 4℃ and seal, and they can be stored for a month.
3.4.2 Standard application solution of sugar: separately pipet 1.00mL, 2.00mL, 3.00mL and 5.00mL of standard stock solution of sugar into a 10mL volumetric flask, add water and scale the volume, and they are equivalent to standard solutions with concentration of 2.0mg/mL, 4.0mg/mL, 6.0mg/mL and 10.0mg/mL.
4 Apparatuses
4.1 Balance: with a sensibility of 0.1mg
4.2 Ultrasonic vibrator
4.3 Magnetic stirrer
4.4 Centrifuge: rotating speed ≥4,000r/min
4.5 High performance liquid chromatograph: with refractive index detector or evaporative light-scattering detector.
4.6 Liquid chromatographic column: amido-chromatographic column with length of 250mm, inner diameter of 4.6mm, film thickness of 5μm, or chromatographic column with equivalent performance.
5 Specimen Preparation and Preservation
5.1 Specimen preparation
5.1.1 Solid sample
Weigh at least 200g of representative sample, pulverize with a grinder, sieve through a 2.0mm round hole screen, mix uniformly, put it into a clean container, seal and mark.
5.1.2 Semi-solid and liquid samples (except for bee honey sample)
Weigh at least 200g (mL) of representative sample, mix sufficiently, put it into a clean container, seal and mark.
5.1.3 Bee honey sample
Stir the uncrystallized sample uniformly with effort; as for the sample with separated crystal, plug the cap of the sample container, put the sample container into water bath (not exceeding 60℃), wait until the sample is dissolved completed, mix well, and rapidly cool to room temperature for subsequent inspection. During dissolving, it shall avoid the water content entering.
5.2 Preservation
Such specimen which deteriorates easily as bee honey is preserved at 0℃~4℃.
6 Analytical Procedures
6.1 Sample treatment
6.1.1 Foods with fat content less than 10%
Weigh 0.5g~10g (to the nearest 0.001g) of pulverized or uniformly-mixed specimen (weigh 10g if sugar content ≤5%; weigh 5g if sugar content is 5%~10%; weigh 2g if sugar content is 10%~40%; and weigh 0.5g if sugar content ≥40%) into a 100mL volumetric flask, add about 50mL of water to dissolve it, add 5mL of zinc acetate solution and 5mL of potassium ferricyanide solution slowly, add water to the scale, carry out magnetic stirring or ultrasonic vibration for 30min, filter with dry filter paper, discard the primary filtrate, then filter the subsequent filtrate through 0.45μm microfiltration membrane or filter the supernatant obtained by centrifugation through 0.45μm microfiltration membrane into the sample container for liquid chromatographic analysis.
6.1.2 Syrup and bee honey
Weigh 1g~2g (to the nearest 0.001g) of uniformly-mixed specimen into a 50mL volumetric flask, add water and scale the volume to 50mL, sufficiently shake well, filter with dry filter paper, discard the primary filtrate, filter the subsequent filtrate through 0.45μm microfiltration membrane or filter the supernatant obtained by centrifugation through 0.45μm microfiltration membrane into the sample container for liquid chromatographic analysis.
6.1.3 Beverage containing carbon dioxide
Pipet uniformly-mixed specimen into the evaporating dish, stir on the water bath (slightly heat) to remove carbon dioxide, pipet 50.0mL into a 100mL volumetric flask, respectively add 5mL of zinc acetate solution and potassium ferricyanide solution slowly, dilute with water to the scale, shake well, leave standstill for 30min, filter with dry filter paper, discard primary filtrate, and filter the subsequent filtrate through 0.45μm microfiltration membrane or filter the supernatant obtained by centrifugation through 0.45μm microfiltration membrane into the sample container for liquid chromatographic analysis.
6.1.4 Foods with fat content greater than 10%
Weigh 5g~10g (to the nearest 0.001g) of pulverized or uniformly-mixed specimen into a 100mL centrifuge tube (with plug), add 50mL of petroleum ether, mix uniformly, exhaust the air, shake for 2min, carry out centrifugation at 1,800 r/min for 15min, and repeat the above-mentioned procedures after removing the petroleum ether until most of the fat is removed. Evaporate the residual petroleum ether, pound the sample with a glass rod, transfer it into a 100mL volumetric flask, rinse the centrifuge tube twice with 50mL of water, add the washing liquid into a 100mL volumetric flask, add 5mL of zinc acetate solution and 5mL of potassium ferricyanide solution slowly, add water to the scale, carry out magnetic stirring or ultrasonic vibration for 30min, filter with filter paper, discard the primary filtrate and filter the subsequent filtrate through 0.45μm microfiltration membrane or filter the supernatant obtained by centrifugation through 0.45μm microfiltration membrane into the sample container for liquid chromatographic analysis.
6.2 Reference conditions for chromatograph
The chromatographic conditions shall meet the requirement that the resolution of fructose, glucose, sucrose, maltose and lactose is greater than 1.5. See Figures A.1 and A.2 in Annex A for chromatograms.
a) Mobile phase: acetonitrile + water = 70+30 (volume ratio);
b) Flow rate of mobile phase: 1.0mL/min;
c) Column temperature: 40℃;
d) Injection volume: 20μL;
e) Condition of refractive index detector: 40℃;
f) Condition of evaporative light-scattering detector: temperature of drift tube: 80℃~90℃; nitrogen pressure: 350kPa; striker: closed.
6.3 Plotting of standard curve
Determine the standard application solution of sugar according to the above-mentioned recommended chromatographic condition, record the peak area or peak height of chromatogram, take the peak area or peak height as longitudinal coordinate and concentration of standard working solution as horizontal ordinate, and linear equation is adopted for refractive index detector; power function equation is adopted for evaporative light-scattering detector to plot the standard curve.
Foreword I
1 Scope
2 Theory
3 Reagents and Materials
4 Apparatuses
5 Specimen Preparation and Preservation
6 Analytical Procedures
7 Expression of Analysis Results
8 Precision
9 Others
10 Theory
11 Reagents and Solutions
12 Apparatuses
13 Specimen Preparation and Preservation
14 Analytical Procedures
15 Expression of Analysis Results
16 Precision
17 Others
Annex A Chromatograms
