GB 5009.279-2016National food safety standard - Determination of Xylitol, Sorbitol, Maltitol and Erythritol in Foods (English PDF)

3.1 Reagents

3.1.1 Acetonitrile (CH3CH): chromatographic purity.

3.1.2 Trichloroacetic acid (CCl3COOH).

3.1.3 Anhydrous sodium carbonate (Na2CO3).

3.2 Preparation of reagents

4.6 Ultrasonic cleaning machine: with a working frequency of 40 kHz and the power of

500 W.

5.1 Sample preparation and pretreatment

5.1.1 Chewing gum

Take at least 20 g of the chewing gum sample; use a blade to cut into small pieces;

put into a closed container to mix. Accurately weigh 2 g of chopped sample; place the sample in a 50 mL centrifuge tube; add 40 mL of water; mix and heat in a water bath at 80°C for 20 min; mix and shake every 6 min; take out and centrifuge at 9 000 r/min for 10 min. Take 8 mL of supernatant in a 10 mL volumetric flask; add water to fix- volume; shake well; filter through a 0.22 µm filter membrane; test on the machine.

5.1.2 Beverage

For non-protein beverages, take at least 200 mL of sample; mix well; place in a closed container. Take 10 mL of beverage in a 50 mL volumetric flask; add water to fix-volume to 50 mL; shake well; filter through a 0.22 µm filter membrane; test on the machine.

For protein beverages, take at least 200 mL of sample; place in a closed container to mix well. Weigh 5 g of sample; place in a 50 mL volumetric flask; add 35 mL of water;

shake well; then sonicate for 30 min; shake it every 6 min; take out and centrifuge at

9 000 r/min for 10 min.

Precipitate protein: add 5 mL of trichloroacetic acid solution (100 g/L) into the supernatant; shake it; then place it at room temperature for 30 min; centrifuge at 9 500 r/min for 10 min. Take 8 mL of the supernatant in a 10 mL volumetric flask; add water to fix-volume. After shaking, take 850 µL of filtrate; add 150 µL of sodium carbonate solution (21.2 g/L); shake and neutralize; or take 10 mL of supernatant; add 20 mL of acetonitrile; shake well and place it at room temperature for 30 min; centrifuge at 9

500 r/min for 10 min; fix-volume the supernatant to 50 mL; shake well; filter through

0.22 µm microporous membrane; test on the machine.

Note: For samples with sugar alcohol content lower than the detection-limit after being precipitated and diluted by acetonitrile, trichloroacetic acid shall be used for precipitation. For samples with low erythritol content (<=1%), acetonitrile shall be used for precipitation. In other cases, both methods are available.

5.1.3 Biscuit, pastry, bread

Take at least 200 g of sample; use a grinder to grind; put it into a closed container to mix. Weigh 1 g ~ 5 g of pulverized sample; place it in a 50 mL centrifuge tube; add 40 mL of water; shake it; then sonicate for 30 min; shake it every 6 min; take out and

10.3 Standard

10.3.1 Xylitol (C5H12O5, CAS No.: 87-99-0), with a purity >= 99%, or a standard substance certified by the state and awarded with a standard substance certificate.

10.3.2 Sorbitol (C6H14O6, CAS No.: 50-70-4), with a purity >= 99%, or a standard substance certified by the state and awarded with a standard substance certificate.

10.3.3 Maltitol (C12H24O11, CAS No.: 585-88-6), with a purity >= 99%, or a standard substance certified by the state and awarded with a standard substance certificate.

10.3.4 Erythritol (C4H10O4, CAS No.: 149-32-6), with a purity >= 99%, or a standard substance certified by the state and awarded with a standard substance certificate.

10.4 Preparation of standard solution

10.4.1 Standard stock solution: respectively and accurately weigh 25 mg of xylitol, 25

mg of sorbitol, 25 mg of maltitol and 35 mg of erythritol, (accurate to 0.1 mg); add water to fix-volume to 10 mL; every milliliter of solution is equivalent to 3.5 mg of erythritol, 2.5 mg of xylitol, 2.5 mg of sorbitol and 2.5 mg of maltitol; it can be stored for 1 month at 4°C if sealed well.

10.4.2 Standard working solution: respectively and accurately transfer 40 µL, 60 µL,

80 µL, 100 µL, 120 µL and 140 µL of various sugar alcohol standard stock solutions;

add water to fix-volume to 1 mL; prepare erythritol into mixed series standard working solution with the concentration of 0.14mg/mL, 0.21 mg/mL, 0.28 mg/mL, 0.35 mg/mL,

0.42 mg/mL and 0.49 mg/mL; and prepare xylitol, sorbitol and maltitol into mixed series standard working solutions with the concentration of 0.10 mg/mL, 0.15 mg/mL, 0.20 mg/mL, 0.25 mg/mL, 0.30 mg/mL and 0.35 mg/mL respectively.

11.1 High performance liquid chromatography: with evaporative light scattering

detector.

11.2 Chromatographic column: amino column with a column length of 250 mm, an

inner diameter of 4.6 mm and a particle size of 5 µm, or equivalent columns.

11.3 Food grinder.

11.4 Analytical balance: with the sensitivity of 0.1 mg and 0.01 g.

11.5 High speed centrifuge: speed >= 9 500 r/min.

11.6 Ultrasonic cleaning machine: with the working frequency of 40 kHz, the power of

500 W.

Precipitate protein: add 5 mL of trichloroacetic acid solution into the supernatant;

shake it; then place it at room temperature for 30 min; centrifuge at 9 500 r/min for 10 min. Take 8 mL of the supernatant in a 10 mL volumetric flask; add water to fix-volume.

After shaking, take 850 µL of filtrate; add 150 µL of sodium carbonate solution; shake and neutralize; or take 10 mL of supernatant; add 20 mL of acetonitrile; shake well and place it at room temperature for 30 min; centrifuge at 9 500 r/min for 10 min; fix-volume the supernatant to 50 mL; shake well. After filtering through a 0.22 µm filter, test it on the machine after dilution.

Note: For samples with sugar alcohol content lower than the detection-limit after being precipitated and diluted by acetonitrile, trichloroacetic acid shall be used for precipitation. For samples with low erythritol content (<=1%), acetonitrile shall be used for precipitation. In other cases, both methods are available.

12.2 Apparatus reference conditions

Apparatus reference conditions are listed as below:

a) Chromatographic column: amino column with a column length of 250 mm, an inner diameter of 4.6 mm and a particle size of 5 µm, or equivalent columns;

b) Column temperature: 30°C;

c) Mobile phase: acetonitrile: water = 80:20;

d) Flow velocity: 1.0 mL/min;

e) Injection volume: 10 µL;

f) Drift tube temperature: 83.5°C;

g) Atomizing gas flow velocity: 2.1 L/min.

12.3 Preparation of the standard curve

Respectively inject 10 µL of standard series working solution into the high performance liquid chromatograph; measure the response value (peak area) of the standard solution under the described chromatographic conditions; use the logarithm to base

10 of the concentration of the standard working solution as the vertical ordinate, the logarithm to base 10 of the response value (peak area) as the abscissa, draw the standard curve.

12.4 Test of sample solution

Inject 10 µL of the sample solution into the high performance liquid chromatograph;

measure the response value (peak area) of the sample under the described chromatographic conditions; determine the sugar alcohol in the sample through the

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