GB 5009.277-2016National food safety standard - Determination of sodium hydrogen diacetate in foods (English PDF)

3.1 Reagents

3.1.1 Phosphoric acid (H3PO4).

3.1.2 Diammonium hydrogen phosphate [(NH4)2HPO4].

3.1.3 Silicone oil [(C2H6OSi)n].

3.2 Preparation of reagents

3.2.1 Phosphoric acid solution (1 mol/L). add 53.5 mL of phosphoric acid into 500 mL

of water; after mixing up, add water to make up to 1,000 mL.

3.2.2 Diammonium hydrogen phosphate (1.5 g/L). weigh 1.5 g of diammonium

hydrogen phosphate; add water to dissolve and make up to 1,000 mL.

3.3 Standard substance

Sodium hydrogen diacetate standard substance [(CH3COO)2HNa, CAS no.. 126-96-5].

purity >= 99%.

3.4 Preparation of standard solutions

3.4.1 Standard stock solution (10 mg/mL). Accurately weigh 1 g (accurate to 0.000 1

g) of sodium hydrogen diacetate standard substance; use water to make up to 100 mL.

The standard stock solution is stored in a refrigerator at 0°C ~ 4°C; the storage life is

3.4.2 Standard working solution. accurately absorb 5.0 mL of standard stock solution

to pour into a volumetric flask of 50 mL; use water to dilute to scale; prepare a standard

working solution of concentration 1.0 mg/mL. The standard working solution is stored

in a refrigerator at 0°C ~ 4°C; the storage life is 1 month.

4.1 High performance liquid chromatograph (HPLC). equipped with an ultraviolet

detector or a diode array detector.

4.2 Analytical balance. sensitivities 0.000 1 g and 0.01 g.

4.3 Blender.

4.4 Steam distillation apparatus. 500 mL.

4.5 Centrifugal machine. rotational speed >= 4,000 r/min.

4.6 Stoppered plastic centrifugal tubes. 50 mL.

4.7 Ultrasonic cleaner.

4.8 pH meter.

4.9 Aqueous phase micropore filter membranes of 0.45 µm.

4.10 Precise pH test paper. pH 0.5 ~ 5.0.

5.1 Sample preparation

In case of solid sample, take 500 g before using a blender to mix up for use; or in case

of liquid sample, shake up for use.

5.2 Sample treatment

5.2.1 The distillation method

Accurately weigh 25 g (accurate to 0.01 g) of sample; place in a distillation flask of 500

mL; add 100 mL of water; use 50 mL of water to rinse the container; transfer to the

distillation flask; add 20 mL of phosphoric acid solution (1 mol/L); 2 drops ~ 3 drops of

silicone oil; carry out steam distillation; place a volumetric flask of 250 mL in the water

bath as the absorption liquid receiver; take out when about 240 mL is distilled; place

aside for 30 min at room temperature; use phosphoric acid solution of 1 mol/L to adjust

the pH to be about 3; add water to make up; shake up; after filtering through aqueous

phase micropore filter membranes of 0.45 µm, carry out liquid chromatographic

determination.

5.2.2 The direct extraction method (apply only to steamed buns and steamed

twisted rolls)

Accurately weigh 5 g (accurate to 0.01 g) of sample to place into a beaker of 100 mL;

add 20 mL of water; add 0.5 mL of phosphoric acid solution of 1 mol/L; mix up; after

ultrasonic extraction for 10 min, use phosphoric acid solution (1 mol/L) to adjust the pH

to about 3; transfer sample to a volumetric flask of 50 mL; use water to make up to

scale; shake up. Transfer all sample into a stoppered plastic centrifugal tube of 50 mL;

carry out centrifugation for 10 min at not lower than 4,000 r/min; take liquid supernatant;

after filtering through aqueous phase micropore filter membranes of 0.45 µm, carry out

liquid chromatographic determination.

5.3 Apparatus reference conditions

a) chromatographic column. C18 column, column length 250 mm, internal diameter

4.6 mm and grain diameter 5 µm, or equivalent;

b) column temperature. 25°C;

c) moving phase. use phosphoric acid solution (1 mol/L) to adjust the pH of

diammonium hydrogen phosphate solution (1.5 g/L) to 2.7 ~ 3.5 (prepare

immediately prior to use) and filter using aqueous phase micropore filter

membranes of 0.45 µm;

d) flow rate. 1.0 mL/min;

e) wavelength. 214 nm;

f) volume of sample. 20 µL;

g) reference conditions for the cleaning of chromatographic column. after test is

completed, use methanol 10% to clean for 1 h; then use methanol 100% to clean

for 1 h.

5.4 Plotting of standard curve

5.4.1 The distillation method

Accurately absorb 1.0 mL, 2.5 mL, 5.0 mL, 7.5 mL, 10.0 mL and 12.5 mL of sodium

hydrogen diacetate standard stock solution to pour into distillation flasks of 500 mL;

other operations are as in 5.2.1. The final concentrations of sodium hydrogen diacetate

standard solution are respectively 0.04 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4

mg/mL and 0.5 mg/mL. Inject them into the liquid chromatograph after filtering through

aqueous phase micropore filter membranes of 0.45 µm; measure the corresponding

peak areas; plot the standard curve using the concentrations of standard working

solutions as the abscissa and the peak areas as the ordinate.

5.4.2 The direct extraction method

Accurately absorb 0.5 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL and 5.0 mL of standard

working solution to pour into volumetric flasks of 10 mL; add 0.2 mL of phosphoric acid

solution (1 mol/L) respectively; use water to make up to 10 mL. The concentrations of

sodium hydrogen diacetate are respectively 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3

mg/mL, 0.4 mg/mL and 0.5 mg/mL. Inject them into the liquid chromatograph after

filtering through aqueous phase micropore filter membranes of 0.45 µm; measure the

corresponding peak areas; plot the standard curve using the concentrations of

standard working solutions as the abscissa and the peak areas as the ordinate.

5.5 Determination of sample solution

Inject sample solution into the liquid chromatograph to obtain the peak area; then

obtain the concentration of sodium hydrogen diacetate in solution to be tested from the

standard curve. For the liquid chromatogram of sodium hydrogen diacetate standard

solution see Figure A.1.

8.1 Limit of detection of the distillation method

When the weight of sample is 25.0 g and the final volume is 250 mL. the limit of

detection of the method is 0.030 g/kg; the limit of quantitation of the method is 0.100

g/kg.

8.2 The direct extraction method

When the weight of sample is 5.0 g and the final volume is 50 mL. the limit of detection

of the method is 0.015 g/kg; the limit of quantitation of the method is 0.05 g/kg.

Annex A

Liquid chromatogram of sodium hydrogen diacetate standard solution

For the liquid chromatogram of sodium hydrogen diacetate standard solution see

Figure A.1.

Figure A.1 – Liquid chromatogram of sodium hydrogen diacetate standard

solution

Ac

eti

c a

cid

Annex B

Method for the determination of acetic acid background in foods

B.1 Analytical procedures

B.1.1 Sample preparation

See 5.1.

B.1.2 Sample treatment (the distillation method)

Accurately weigh 25 g (accurate to 0.01 g) of sample; place in a distillation flask of 500

mL; add 100 mL of water; use 50 mL of water to rinse the container; transfer to the

distillation flask; 2 drops ~ 3 drops of silicone oil; carry out steam distillation; place a

volumetric flask of 250 mL in the water bath as the absorption liquid receiver; take out

when about 240 mL is distilled; place aside for 30 min at room temperature; use

phosphoric acid solution of 1 mol/L to adjust the pH to be about 3; add water to make

up; shake up; after filtering through aqueous phase micropore filter membranes of 0.45

µm, carry out liquid chromatographic determination.

B.1.3 Apparatus reference conditions

See 5.3.

B.1.4 Plotting of standard curve

See 5.4.1.

B.1.5 Determination of sample solution

See 5.5.

B.2 Expression of analytical results

The content of acetic acid background in sample is calculated in accordance with

Equation (2).

where.

X1 – the content of acetic acid background in sample, in g/kg;

rho1 – the concentration of acetic acid in sample solution which is obtained from the

standard curve, in mg/mL;

V1 – the final volume of sample solution made up to, in mL;

1,000 – the conversion coefficient;

m1 – the mass of sample, in g;

f1 – the dilution ratio.

B.3 Precision

See Clause 7.

......