GB 5009.8-2023 PDF in English
GB 5009.8-2023 (GB5009.8-2023) PDF English
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National food safety standard - Determination of fructose, glucose, sucrose, maltose and lactose in foods
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Standards related to: GB 5009.8-2023
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GB 5009.8-2023: PDF in English GB 5009.8-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Determination of fructose,
glucose, sucrose, maltose and lactose in foods
ISSUED ON. SEPTEMBER 06, 2023
IMPLEMENTED ON. MARCH 06, 2024
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
2 Principle... 4
3 Reagents and materials... 4
4 Instruments and apparatuses... 6
5 Analysis steps... 6
6 Expression of analysis results... 8
7 Precision... 9
8 Others... 9
9 Principle... 9
10 Reagents and materials... 9
11 Instruments and apparatuses... 11
12 Test steps... 11
13 Expression of analysis results... 14
14 Precision... 14
15 Others... 14
16 Principle... 15
17 Reagents and solutions... 15
18 Instruments and apparatuses... 16
19 Analysis steps... 16
20 Expression of analysis results... 19
21 Precision... 21
22 Principle... 21
23 Reagents and materials... 21
24 Instruments and apparatuses... 22
25 Analysis steps... 22
26 Expression of analysis results... 24
27 Precision... 25
Appendix A HPLC chromatograms of standard solutions of fructose, glucose, sucrose,
maltose and lactose... 26
Appendix B Ion chromatogram of standard solutions of fructose, glucose, sucrose,
maltose and lactose... 28
National food safety standard
Determination of fructose, glucose, sucrose, maltose and
lactose in foods
1 Scope
This Standard specifies the methods for the determination of fructose, glucose, sucrose,
maltose and lactose in foods.
Method 1 - High performance liquid chromatography applies to the determination of
fructose, glucose, sucrose, maltose and lactose in grains and grain products, milk and
dairy products, fruits, vegetables and fruit and vegetable products, sweeteners, candies,
beverages and infant foods.
Method 2 - Ion chromatography applies to the determination of fructose, glucose,
sucrose, maltose, and lactose in foods.
Method 3 - Acid hydrolysis-Rhein-Eynon method applies to the determination of
sucrose in foods.
Method 4 - Rhine-Enon method applies to the determination of lactose in infant foods
and dairy products.
Method 1 - High performance liquid chromatography
2 Principle
The fructose, glucose, sucrose, maltose and lactose in the sample are extracted, and then
separated by high performance liquid chromatography column, detected by a
differential refractive index detector or an evaporative light scattering detector, and
quantified by the external standard method.
3 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical reagents, and
the water is grade-1 water specified by GB/T 6682.
3.1 Reagents
3.1.1 Acetonitrile (C2H3N). chromatographic purity.
3.1.2 Zinc acetate [Zn(CH3COO)2·2H2O].
3.1.3 Potassium ferrocyanide [K4Fe(CN)6·3H2O].
3.1.4 Glacial acetic acid (CH3COOH).
3.2 Preparation of reagents
3.2.1 Zinc acetate solution (1 mol/L). Weigh 21.9 g of zinc acetate; add 3 mL of glacial
acetic acid; dissolve in water and dilute to 100 mL; mix well.
3.2.2 Potassium ferrocyanide solution (0.25 mol/L). Weigh 10.6 g of potassium
ferrocyanide; dissolve in water and dilute to 100 mL; mix well.
3.3 Standards
3.3.1 Fructose (C6H12O6, CAS number. 57-48-7). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
3.3.2 Glucose (C6H12O6, CAS number. 50-99-7). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
3.3.3 Sucrose (C12H22O11, CAS number. 57-50-1). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
3.3.4 Maltose (C12H22O11, CAS number. 69-79-4). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
3.3.5 Lactose (C12H22O11, CAS number. 63-42-3). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
3.4 Preparation of standard solutions
3.4.1 Mixed standard stock solution (20.0 mg/mL). Respectively weigh 1 g (accurate
to 0.001 g) each of fructose dried at 90 °C ± 2 °C for 2 h and glucose, sucrose, maltose
and lactose dried at 96 °C ± 2 °C for 2 h; dissolve in water and then transfer to the 50
mL volumetric flasks; add 2.5 mL of acetonitrile; use water to adjust the volume to the
mark. Store at 0 °C ~ 4 °C and seal, with a shelf life of 3 months.
3.4.2 Mixed standard working solution. Take 0.100 mL, 1.00 mL, 2.00 mL, 3.00 mL
and 5.00 mL of mixed standard stock solution (20.0 mg/mL) into the 10.0 mL
volumetric flask; use water to dilute to the mark, to prepare the mixed standard working
solutions of fructose, glucose, sucrose, maltose and lactose with mass concentrations of
0.200 mg/mL, 2.00 mg/mL, 4.00 mg/mL, 6.00 mg/mL and 10.0 mg/mL. The
concentration of the mixed standard working solution can be appropriately adjusted
according to the concentration of the actual sample solution. Prepare when necessary.
3.5 Materials
Weigh 50 g (accurate to 0.01 g) of the mixed sample in an evaporating dish; stir gently
on a water bath to remove gas and alcohol; transfer to a 100 mL volumetric flask after
cooling; slowly add 5 mL of zinc acetate solution and 5 mL of potassium ferrocyanide
solution; use water to adjust the volume to the mark; mix well; let stand.
5.1.2.3 Syrup and honey samples
Weigh 1 g ~ 2 g (accurate to 0.001 g) of the mixed sample into a 100 mL colorimetric
tube; add about 50 mL of water; vortex and mix until fully dissolved; transfer to a 100
mL volumetric flask; use water to fix the volume to the mark; mix well; let stand.
5.1.2.4 Other samples
Weigh 1 g ~ 10 g (accurate to 0.001 g) of the crushed or mixed sample (10 g when the
target sugar content is ≤5%; 5 g when the content is 5% ~ 10%; 2 g when the content is
10% ~ 40%; 1 g when the content is ≥40%) into a 100 mL colorimetric tube; add about
50 mL of water; then, slowly add 5 mL of zinc acetate solution and 5 mL of potassium
ferrocyanide solution; vortex and mix, and sonicate for 30 minutes; transfer to a 100
mL volumetric flask; use water to adjust the volume to the mark; mix well; let stand.
5.1.3 Purification
For the above sample extract solution, use a filter paper to filter (discard the primary
filtrate) or centrifuge to obtain the supernatant; then, use a 0.45 μm water-based
membrane syringe to filter into a sample bottle for analysis by a high-performance
liquid chromatograph.
5.2 Apparatus reference conditions
Apparatus reference conditions are as below.
a) Chromatographic column. Amino chromatographic column (4.6 mm × 250 mm,
particle size 5 μm, aminosilane bonded silica gel as filler), or one with equivalent
performance;
b) Mobile phase. acetonitrile + water = 70 + 30 (volume ratio);
c) Flow velocity. 1.0 mL/min;
d) Column temperature. 40 ℃;
e) Injection volume. 10 μL;
f) Differential refractive index detector conditions. temperature 40 °C;
g) Evaporative light scattering detector conditions. drift tube temperature 80 °C ~
90 °C; nitrogen flow rate 2.5 L/min.
5.3 Preparation of the standard curve
Inject the mixed standard working solution into the high-performance liquid
chromatograph in order from low to high concentration, and measure the corresponding
peak areas or peak heights of fructose, glucose, sucrose, maltose and lactose. For the
differential refractive index detector, use the concentration of the standard working fluid
as the abscissa and the peak area or peak height as the ordinate to draw a standard curve;
for the evaporative light scattering detector, use the power function of the concentration
of the standard working solution as the abscissa and the power function of the peak area
or peak height as the ordinate to draw a standard curve. Refer to Appendix A for HPLC
chromatograms of standard solutions of fructose, glucose, sucrose, maltose and lactose.
5.4 Determination of sample solution
Inject the sample solution into the high-performance liquid chromatograph;
qualitatively record the peak area or peak height of the target substance based on the
retention time; obtain the concentrations of fructose, glucose, sucrose, maltose and
lactose in the sample solution based on the standard curve.
5.5 Blank test
Except that no sample is added, proceed according to the above steps.
6 Expression of analysis results
Calculate the contents of fructose, glucose, sucrose, maltose and lactose in the sample
according to Formula (1).
Where.
X – the content of fructose, glucose, sucrose, maltose and lactose in the sample, in
grams per hundred grams (g/100 g);
ρ – the mass concentration of fructose, glucose, sucrose, maltose and lactose in the
sample solution obtained according to the standard curve, in milligrams per
milliliter (mg/mL);
ρ0 – the mass concentration of fructose, glucose, sucrose, maltose and lactose in the
blank obtained according to the standard curve, in milligrams per milliliter
(mg/mL);
V – constant volume, in milliliters (mL);
f – dilution factor;
10.2.2 Acetic acid solution (3%, volume fraction). Measure 3 mL of glacial acetic acid;
use water to dilute to 100 mL; mix well.
10.3 Standards
10.3.1 Fructose (C6H12O6, CAS number. 57-48-7). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
10.3.2 Glucose (C6H12O6, CAS number. 50-99-7). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
10.3.3 Sucrose (C12H22O11, CAS number. 57-50-1). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
10.3.4 Maltose (C12H22O11, CAS number. 69-79-4). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
10.3.5 Lactose (C12H22O11, CAS number. 63-42-3). purity ≥99%, or standard material
certified by the country and awarded a standard material certificate.
10.4 Preparation of standard solution
10.4.1 Mixed standard stock solution (10.0 mg/mL). Weigh 1 g (accurate to 0.001 g)
each of fructose dried at 90 °C ± 2 °C for 2 h and glucose, sucrose, maltose and lactose
dried at 96 °C ± 2 °C for 2 h; add water to dissolve and transfer to a 100 mL volumetric
flask; add 2 mL of acetic acid solution; use water to dilute to the mark. Store at 0 °C ~
4 °C and seal, with a shelf life of 3 months.
10.4.2 Mixed standard intermediate solution (100 mg/L). Pipette 1.00 mL of fructose,
glucose, sucrose, maltose and lactose mixed standard stock solution (10.0 mg/mL) into
the 100 mL volumetric flask; use water to dilute to the mark. Store at 0 °C ~ 4 °C and
seal, with a shelf life of 1 months.
10.4.3 Mixed standard working solution. Respectively take 0.250 mL, 0.500 mL, 1.00
mL, 2.00 mL and 2.50 mL of the mixed standard intermediate solution (100 mg/L) into
the 10 mL volumetric flasks; use water to dilute to the mark, to prepare the mixed
standard working solutions of fructose, glucose, sucrose, maltose and lactose with mass
concentrations of 2.50 mg/L, 5.00 mg/L, 10.0 mg/L, 20.0 mg/L and 25.0 mg/L. The
concentration of the mixed standard working solution can be appropriately adjusted
according to the concentration of the actual sample solution. Prepare when necessary.
10.5 Materials
10.5.1 0.45 μm water-based membrane syringe filter (except cellulose membrane).
10.5.2 Purification column. C18 solid-phase extraction cartridge (1.0 mL) or one with
equivalent performance.
10.5.3 Syringe.
11 Instruments and apparatuses
11.1 Ion chromatograph. equipped with pulse ampere detector.
11.2 Sample crushing equipment. high-speed crusher.
11.3 Ultrasonic cleaner.
11.4 Analytical balance. sensitivity 1 mg.
11.5 Vortex mixer.
11.6 Centrifuge. speed ≥ 4 000 r/min.
11.7 Constant-temperature drying oven.
11.8 Constant-temperature water bath device.
12 Test steps
12.1 Sample pretreatment
12.1.1 Sample preparation
Take an appropriate amount of representative sample; for drinks and other liquid
homogeneous samples, shake directly; for non-uniform samples, homogenize or crush
evenly; for frozen drinks, melt at room temperature, stir thoroughly and, if necessary,
heat and stir in a water batch at 30 °C ~ 40 °C; for sauces, grind or homogeneously mix;
for chocolate, heat and melt in a water bath at 50 °C ~ 60 °C, and stir thoroughly while
it is hot.
12.1.2 Sample extraction
12.1.2.1 Insoluble samples such as gum-based candies and chocolates
Accurately weigh 2 g (accurate to 0.001 g) of the sample into a 100 mL colorimetric
tube; add about 50 mL of 50 °C ~ 60 °C hot water; vortex or stir, until the sample is
fully dissolved; add 2 mL of acetic acid solution; vortex to mix, and sonicate for 30 min;
transfer to a 100 mL volumetric flask and use water to adjust the volume to the mark;
mix well; let stand for 20 minutes.
12.1.2.2 Syrup and honey samples
Weigh 2 g (accurate to 0.001 g) of the mixed sample into a 100 mL colorimetric tube;
add about 50 mL of water; vortex and mix until fully dissolved; transfer to a 100 mL
volumetric flask; use water to adjust the volume to the mark; mix well; let stand for 20
minutes.
12.1.2.3 Samples containing gas and alcohol
Accurately weigh 10 g of the sample (accurate to 0.001 g) in an evaporating dish; stir
gently on a water bath to remove gas and alcohol; after cooling, transfer it to a 100 mL
volumetric flask with water; add 2 mL of acetic acid solution; use water to adjust the
volume to the mark; mix well; let stand for 20 minutes.
12.1.2.4 Other samples
Weigh 2 g (accurate to 0.001 g) of solid sample and 5 g ~ 10 g (accurate to 0.001 g) of
semi-solid or liquid sample into a 100 mL colorimetric tube; add about 50 mL of water;
vortex to mix; then, add 2 mL of acetic acid solution; after mixing, sonicate for 30
minutes; transfer to a 100 mL volumetric flask and use water to dilute to the mark; mix
well; let stand for 20 minutes.
12.1.3 Sample purification
The sample extraction solution can be diluted by an appropriate multiple according to
the target sugar content in the sample. When detecting lactose in infant formula milk
powder, dilute it 1 000 times and then purify it; when detecting high sugar content in
honey and candy, dilute it 500 times and then purify it.
After filtering or centrifuging the above sample extraction solution or dilution solution
to obtain the supernatant, pass it through a 0.45 μm aqueous membrane syringe filter
and a C18 solid phase extraction cartridge (1.0 mL) in sequence; discard the first 3 mL;
collect subsequent eluates for testing.
Before use, pass 10 mL of methanol and 15 mL of water in sequence through the C18
solid-phase extraction cartridge (1.0 mL); let stand to activate for 30 min.
12.2 Apparatus reference conditions
Apparatus reference conditions are as below.
a) Chromatographic column. anion exchange column (4 mm × 250 mm, particle size
10 μm, quaternary ammonium salt as functional group,
polystyrene/divinylbenzene polymer resin as filler) (with guard column 4 mm ×
50 mm), or one with equivalent performance;
b) Flow velocity. 1.0 mL/min;
c) Injection volume. 10 μL;
d) Pulse ampere detector. Au working electrode; see Table 1 for sugar detection
waveform reference conditions;
16 Principle
After the protein is removed from the sample, the sucrose is hydrolyzed by hydrochloric
acid and converted into reducing sugar, and is measured as reducing sugar. The
difference before and after hydrolysis multiplied by the corresponding coefficient is the
sucrose content. Raffinose, stachyose, galactooligosaccharides, fructans, polydextrose
and resistant dextrin will interfere with the determination of sucrose.
17 Reagents and solutions
Unless otherwise specified, all the reagents in this method are analytical reagents, and
the water is grade-3 water specified by GB/T 6682.
17.1 Reagents
17.1.1 Zinc acetate [Zn(CH3COO)2·2H2O].
17.1.2 Potassium ferrocyanide [K4Fe(CN)6·3H2O].
17.1.3 Hydrochloric acid (HCl).
17.1.4 Sodium hydroxide (NaOH).
17.1.5 Methyl red (C15H15N3O2). indicator agent.
17.1.6 Methylene blue (C16H18ClN3S·3H2O). indicator agent.
17.1.7 Copper sulfate (CuSO4·5H2O).
17.1.8 Potassium sodium tartrate (C4H4O6KNa·4H2O).
17.1.9 Glacial acetic acid (CH3COOH).
17.1.10 Ethanol (C2H5OH). 95%.
17.2 Preparation of reagents
17.2.1 Zinc acetate solution (1 mol/L). Weigh 21.9 g of zinc acetate; add 3 mL of glacial
acetic acid; dissolve in water and dilute to 100 mL; mix well.
17.2.2 Potassium ferrocyanide solution (0.25 mol/L). Weigh 10.6 g of potassium
ferrocyanide; dissolve in water and dilute to 100 mL; mix well.
17.2.3 Hydrochloric acid solution (50%, volume fraction). Weigh 50 mL of
hydrochloric acid; slowly add 50 mL of water; let it cool; mix well.
17.2.4 Sodium hydroxide solution (40 g/L). Weigh 4.0 g of sodium hydroxide; add
water to dissolve; cool; use water to dilute to 100 mL; mix well.
17.2.5 Methyl red indicator solution (1 g/L). Weigh 0.1 g of methyl red; use 95%
ethanol to dissolve and dilute to 100 mL; mix well.
17.2.6 Sodium hydroxide solution (200 g/L). Weigh 20.0 g of sodium hydroxide; add
water to dissolve; cool; use water to dilute to 100 mL; mix well.
17.2.7 Alkaline cupric tartrate solution A. Weigh 15.0 g of copper sulfate and 0.05 g of
methylene blue; dissolve in water and dilute to 1 000 mL; mix well.
17.2.8 Alkaline cupric tartrate solution B. Weigh 50.0 g of sodium potassium tartrate
and 75.0 g of sodium hydroxide; dissolve in water; then, add 4.0 g of potassium
ferrocyanide; after complete dissolution, use water to dilute to 1 000 mL; mix well;
store in a glass bottle with rubber stopper.
17.3 Standard
Glucose (C6H12O6, CAS number. 50-99-7). purity ≥99%, or standard material certified
by the country and awarded a standard material certificate.
17.4 Preparation of standard solutions
Glucose standard solution (1.00 mg/mL). Weigh 1 g (accurate to 0.001 g) of glucose
that has been dried in an oven at 96 °C ± 2 °C for 2 hours; add water to dissolve; transfer
to a 1 000 mL volumetric flask; add 5 mL of hydrochloric acid; use water to adjust the
volume to the mark. Store in a sealed container at 0 °C ~ 4 °C.
18 Instruments and apparatuses
18.1 Analytical balance. the sensitivity is 1 mg and 10 mg.
18.2 Constant-temperature water bath device.
18.3 Adjustable-temperature electric stove.
18.4 Acid burette. 25 mL.
18.5 Sample crushing equipment. high-speed crusher.
18.6 Constant-temperature drying oven.
19 Analysis steps
19.1 Sample pretreatment
19.2 Acid hydrolysis
Take 2 portions of the sample treatment solution, 50.0 mL each, and place them in the
100 mL volumetric flasks, respectively.
19.2.1 Before transformation. Use water to dilute 1 portion to the mark and mix well.
19.2.2 After transformation. Add 5 mL of hydrochloric acid solution to 1 portion; heat
in a water bath at 68 °C ~ 70 °C for 15 minutes; add 2 drops of methyl red indicator
solution after cooling; use sodium hydroxide solution (200 g/L) to neutralize to
neutrality; use water to adjust the volume to the mark; mix well.
19.3 Calibration of alkaline cupric tartrate solution
Take 5.0 mL of alkaline cupric tartrate solution A and 5.0 mL of alkaline cupric tartrate
solution B into a 150 mL Erlenmeyer flask; add 10 mL of water; add 2 ~ 4 glass beads;
add about 9 mL of glucose standard solution from the burette; control the heating-to-
boiling process within 2 minutes; add glucose standard solution dropwise at a rate of 1
drop/2 seconds while it is hot, until the blue color of the solution just fades away as the
end point; record the total volume of glucose standard solution consumed; operate 3
portions in parallel at the same time, and take the average value. Calculate the mass A
(mg) of glucose equivalent to each 10 mL of alkaline cupric tartrate solution (5 mL each
of alkaline tartaric acid solutions A and B).
Note. 4 mL ~ 20 mL of alkaline cupric tartrate solution (half-and-half solutions A and
B) can also be calibrated according to the above method to adapt to the
concentration changes of reducing sugars in the sample.
19.4 Determination of sample solution
19.4.1 Predictive titration. Take 5.0 mL of alkaline cupric tartrate solution A and 5.0
mL of alkaline cupric tartrate solution B into a 150 mL Erlenmeyer flask; add 10 mL of
distilled water; add 2 ~ 4 glass beads; heat on an electric furnace; let it boil within 2
minutes; maintain the boiling state for 15 seconds; drop in the sample solution before
transformation (19.2.1) or the sample solution after transformation (19.2.2) until the
blue color of the solution just fades away, which is the end point; read the volume of
the sample solution used.
19.4.2 Accurate titration. Take 5.0 mL of alkaline cupric tartrate solution A and 5.0 mL
of alkaline cupric tartrate solution B into a 150 mL Erlenmeyer flask; add 10 mL of
distilled water; add 2 ~ 4 glass beads; release the sample solution 1 mL less than the
predicted volume of the predictive titration (19.4.1) from the burette; place it on the
electric stove and let it boil within 2 minutes; maintain the boiling state for 2 minutes;
slowly add the sample solution at a rate of 1 drop/2 seconds until the blue color of the
solution just fades as the end point; record the volume (V) of the sample solution
consumed.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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