GB 5009.204-2025National food safety standard - Determination of acrylamide in food (English PDF)

1 Scope

1 The document lays down the methods for determining acrylamide in food.

1 The first method, liquid chromatography-tandem mass spectrometry, applies to the determination of acrylamide in food.

1 The second method, gas chromatography-mass spectrometry, applies to the determination of acrylamide in food other than coffee, tea, sugar, infant formula food and complementary food for infants and young children.

2 Principle of the first method

2 The acrylamide in the test sample is extracted with water, cleaned up by dispersive solid phase extraction, by a solid phase extraction cartridge or by a packed chromatographic column, and measured by liquid chromatography-tandem mass spectrometry with quantification by the internal standard method.

3 Reagents and materials of the first method

3 Unless otherwise stated the reagents are of analytical grade and the water is grade one water as defined in GB/T 6682.

3.1 The reagents are formic acid, methanol and acetonitrile of chromatographic grade, together with n-hexane, ethyl acetate, anhydrous sodium sulfate, ammonium sulfate, sodium chloride and dry ice.

3.2 A saturated ammonium sulfate solution and a 0.1% formic acid solution in water are prepared, both immediately before use.

3.3 The standards are acrylamide, CAS number 79-06-1, of purity not less than 99%, or a nationally certified standard issued with a reference material certificate, and carbon-13 labelled acrylamide, CAS number 287399-26-2, of purity not less than 98%.

3.4 An acrylamide stock standard solution of 1000 mg/L in methanol is prepared and kept at -18 °C protected from light for up to six months; an intermediate solution of 100 mg/L in methanol is valid for three months; working solutions I and II of 10 mg/L and 1 mg/L in water are prepared immediately before use. The internal standard stock solution of 1000 mg/L in methanol is likewise valid for six months and its working solution of 10 mg/L in water is prepared immediately before use. The series of working standard solutions is made up in water from working solutions I and II with the addition of the internal standard working solution, giving acrylamide concentrations of 10, 50, 100, 200, 500 and 1000 micrograms per litre and an internal standard concentration of 100 micrograms per litre; the concentrations may be adjusted to suit the samples.

3.5 The materials are granular diatomaceous earth; a dispersive clean-up tube containing graphitised carbon black, an octadecyl sorbent, ethylenediamine-N-propylsilane, a strong cation exchange material and magnesium sulfate, or an equivalent; a hydrophilic-lipophilic balanced solid phase extraction cartridge of 6 mL capacity holding 200 mg of a divinylbenzene and N-vinylpyrrolidone copolymer, or an equivalent; a mixed-mode silica bonded cartridge of 3 mL capacity holding 200 mg of a mixed strong cation and strong anion exchange bonded phase, or an equivalent; a glass chromatographic column 30 cm long and 1.8 cm in internal diameter; and aqueous microporous filter membranes of 0.22 micrometre and 0.45 micrometre pore size.

4 Instruments and equipment of the first method

4 The equipment is a liquid chromatograph-tandem mass spectrometer fitted with an electrospray ionisation source; a tissue grinder; a rotary evaporator; a nitrogen evaporator; a shaker; a vortex mixer; an electronic balance with graduations of 0.01 mg and 1 mg; and a centrifuge reaching at least 10000 r/min.

5 Analytical procedure of the first method

5.1 For baked foods, puffed foods and fried cereal foods, 100 g of sample is ground with 100 g of dry ice in a tissue grinder and stored at -18 °C once the dry ice has fully sublimed. A note warns that dry ice is extremely cold and is to be handled with thick cotton gloves or tongs, that the carbon dioxide released as it sublimes raises the pressure inside the grinder, so that the pressure rating of the chamber is to be respected to prevent it bursting, and that the room is to be well ventilated. For coffee, tea, sugar, infant formula food and complementary food for infants and young children, 100 g of sample is ground in a tissue grinder, instant coffee and infant milk powder being sampled directly, and stored at -18 °C; a note asks that overheating during grinding be avoided.

5.2 For extraction, 0.5 g to 2 g of sample is weighed to the nearest 0.001 g, the internal standard working solution is added, 10 microlitres for the packed column method and 20 microlitres for the dispersive and cartridge methods, and 10 mL of water is added. The mixture is shaken for 30 min and centrifuged at 4000 r/min for 10 min, and the supernatant is taken for clean-up. Where no supernatant can be obtained by centrifugation, as with instant coffee or infant milk powder, the suspension is taken instead.

5.3.1 In the dispersive solid phase extraction clean-up, which applies to all foods, the supernatant is extracted twice with 5 mL of n-hexane with centrifugation at 10000 r/min, the organic phase being discarded each time; 10 mL of acetonitrile is added and the mixture is extracted for 10 min, 0.5 g of sodium chloride and 4 g of anhydrous sodium sulfate are added as salting-out agents and the mixture is shaken for 5 min and centrifuged; 2.00 mL of the supernatant is vortexed for 5 min with the dispersive clean-up tube and centrifuged, and 1 mL of the resulting supernatant is concentrated almost to dryness under nitrogen, redissolved in 0.20 mL of water and filtered through a 0.22 micrometre aqueous membrane before measurement.

5.3.2 The other clean-up methods apply to foods other than coffee, tea, sugar, infant formula food and complementary food for infants and young children. In the cartridge method the supernatant is extracted twice with n-hexane, filtered through a 0.45 micrometre membrane, passed through a hydrophilic-lipophilic balanced cartridge conditioned with methanol and water and eluted with 4 mL of 80% aqueous methanol, and the combined effluent and eluate are passed under gravity through a mixed-mode bonded cartridge likewise conditioned with methanol and water; the whole effluent is concentrated almost to dryness under nitrogen, made up to 1.00 mL with water and filtered through a 0.22 micrometre membrane. In the packed column method 15 g of ammonium sulfate is dissolved in the supernatant, the mixture is shaken for 10 min and centrifuged, and 10 mL of the supernatant, made up if necessary with saturated ammonium sulfate solution, is mixed with 5 g of diatomaceous earth and charged onto a glass column packed with glass wool, 10 g of anhydrous sodium sulfate and 2 g of diatomaceous earth; the column is washed with 70 mL of n-hexane, which is discarded, and eluted with 70 mL of ethyl acetate at a flow rate of 2 mL/min, and the eluate is evaporated almost to dryness under reduced pressure in a water bath at 45 °C, the residue being washed over with three 1 mL portions of ethyl acetate into a tube containing 1.00 mL of water; after the organic layer has been blown off under nitrogen, 1 mL of n-hexane is added, the tube is vortexed and centrifuged, and the lower aqueous phase is filtered through a 0.22 micrometre membrane before measurement.

5.4 The reference chromatographic conditions are a C18 column of 3.0 mm by 150 mm and 1.8 micrometre particle size, with a stated carbon load of 9%, a pore size of 9.5 nm, double end-capping and a specific surface area of 160 m2/g, or an equivalent, with a matching guard column; mobile phase A of 0.1% formic acid in water and mobile phase B of methanol, run under the gradient of Table 1; a flow rate of 0.2 mL/min; an injection volume of 10 microlitres; and ambient column temperature. The gradient of Table 1 holds 98% A and 2% B from 0.0 min to 2.0 min, changes to 90% A and 10% B by 4.0 min and holds to 6.0 min, changes to 5% A and 95% B by 7.0 min and holds to 8.5 min, and returns to 98% A and 2% B by 8.6 min, held to 10.0 min.

5.4.2 The reference mass spectrometric conditions are multiple reaction monitoring; positive electrospray ionisation; a capillary voltage of 2500 V; an ion source temperature of 150 °C; a desolvation gas temperature of 600 °C; a cone gas flow of 150 L/h; a nebuliser gas pressure of 700 kPa; and a collision gas flow of 0.15 mL/min. The ion pairs, declustering voltages and collision energies are given in Table 2, whose rows the extracted text runs together so that the individual figures cannot be assigned to their columns; the quantifier transitions are named in the body of the document as m/z 72 greater than 55 for acrylamide and m/z 72 greater than 58 for the carbon-13 labelled internal standard.

5.5 The calibration curve is drawn by injecting the series of working standard solutions and plotting the ratio of the peak areas of acrylamide and of its internal standard, taken from the quantifier ion mass chromatograms, against the acrylamide concentration in micrograms per litre. The reference mass chromatogram is Figure A.1 of Annex A.

5.6 For qualitative determination, the retention time of the analyte peak in the sample is to agree with that in the corresponding standard solution to within plus or minus 2.5%, both the quantifier and the qualifier ions are to appear, and the relative abundance ratio of the qualifier to the quantifier ion is to agree with that of a standard solution of comparable concentration within the deviations of Table 3, namely plus or minus 20% where the relative abundance is greater than 50%, plus or minus 25% where it is greater than 20% and not greater than 50%, plus or minus 30% where it is greater than 10% and not greater than 20%, and plus or minus 50% where it is not greater than 10%.

5.7 For quantitative determination the ratio of the peak areas of acrylamide at m/z 72 greater than 55 and of the internal standard at m/z 72 greater than 58 is measured and the concentration is read from the calibration curve. The response is to lie within the range of the curve; if the linear range is exceeded, the test portion or the range of the curve is adjusted and the measurement repeated.

5.8 A blank test is carried out through the whole procedure without a test portion. If the analyte concentration in the blank solution presented to the instrument is above the concentration corresponding to the quantification limit of the method, the reagents or the glassware are to be changed until the background falls below the quantification limit.

6 Expression of results

6 The acrylamide content of the sample is calculated by formula (1), whose terms are the acrylamide concentration in micrograms per litre corresponding to the ratio of the peak areas of acrylamide at m/z 72 greater than 55 and of the internal standard at m/z 72 greater than 58, the volume of the sample extract, the final volume after clean-up, the unit conversion factors, the test portion mass and the volume of the sample solution taken for clean-up. The result is expressed in micrograms per kilogram.

6 Results of 100 or less are reported to three significant figures and results above 100 to one decimal place.

6 A note states that for the dispersive solid phase extraction method the volume of the sample extract is taken as the 10 mL of acetonitrile added and the volume taken for clean-up as 1 mL.

7 Precision of the first method

7 The absolute difference between two independent results obtained under repeatability conditions is not to exceed 20% of their arithmetic mean.

8 Other

8 For a test portion of 1 g the detection limit of the method is 3 micrograms per kilogram and the quantification limit 10 micrograms per kilogram.

9 Principle of the second method

9 The acrylamide in the test sample is extracted with water, cleaned up on a packed column, derivatised with a bromine reagent and measured by gas chromatography-mass spectrometry with quantification by the internal standard method.

10 Reagents and materials of the second method

10 Unless otherwise stated the reagents are of analytical grade and the water is grade one water as defined in GB/T 6682.

10.1 The reagents are n-hexane, ethyl acetate, anhydrous sodium sulfate baked for 4 h at 400 °C, ammonium sulfate, sodium thiosulfate pentahydrate, bromine, hydrobromic acid of content above 48.0% and potassium bromide.

10.2 Saturated bromine water is prepared by adding 8 mL of bromine to 100 mL of water in a brown bottle kept at 4 °C in the dark for 8 h and taking the upper layer. The bromine reagent is prepared from 20.0 g of potassium bromide, 1.0 mL of hydrobromic acid and 16.0 mL of saturated bromine water, made up to 100 mL and kept at 4 °C in the dark. A sodium thiosulfate solution of 0.1 mol/L and a saturated ammonium sulfate solution are likewise prepared immediately before use.

10.3 The standards are the same acrylamide and carbon-13 labelled acrylamide as in the first method.

10.4 The acrylamide and internal standard solutions are those of the first method. The series of working standard solutions is made up in water with the internal standard, giving acrylamide concentrations of 10, 50, 200, 500 and 1000 micrograms per litre and an internal standard concentration of 100 micrograms per litre; the concentrations may be adjusted to suit the samples.

10.5 The materials are granular diatomaceous earth and a glass chromatographic column 30 cm long and 1.8 cm in internal diameter.

11 Instruments and equipment of the second method

11 The equipment is a gas chromatograph-quadrupole mass spectrometer; a tissue grinder; a rotary evaporator; a nitrogen evaporator; a shaker; a vortex mixer; an electronic balance with graduations of 0.01 mg and 1 mg; and a centrifuge reaching at least 10000 r/min.

12 Analytical procedure of the second method

12.1 The test samples are prepared as in 5.1.

12.2 For extraction, 2 g of sample is weighed to the nearest 0.001 g, 10 microlitres of the internal standard solution and 10 mL of water are added, the mixture is shaken for 30 min and centrifuged at 4000 r/min for 10 min, and the supernatant is kept.

12.3 The clean-up follows the packed column procedure of the first method, ending with the lower aqueous phase, which is kept for derivatisation.

12.4 For derivatisation, 1 mL of the bromine reagent is added to the sample extract, the tube is vortexed and left at 4 °C for at least 1 h, and about 100 microlitres of the 0.1 mol/L sodium thiosulfate solution is added with vortexing to destroy the remaining derivatising agent. The mixture is extracted twice with 2 mL of ethyl acetate with vortexing for 1 min and centrifugation at 4000 r/min for 5 min, the upper organic phases being combined over 0.1 g of anhydrous sodium sulfate, left to stand for at least 0.5 h, transferred to another tube, blown almost to dryness under nitrogen and taken up in 0.1 mL of ethyl acetate for measurement. Aliquots of 1.0 mL of each standard solution in the series are derivatised in the same way.

12.5 The reference chromatographic conditions are a weakly polar capillary column of 5% phenyl methyl polysiloxane 30 m long, 0.25 mm in internal diameter with a film thickness of 0.25 micrometre, or an equivalent; an inlet programme holding 120 °C for 2 min and rising at 40 °C/min to 240 °C, held for 5 min; an oven programme holding 65 °C for 1 min, rising at 15 °C/min to 200 °C and then at 40 °C/min to 240 °C, held for 5 min; high purity helium carrier gas of purity above 99.999%, the head pressure being stated in the document as 69 mPa, equivalent to 10 psi; and splitless injection of 1 microlitre. The reference mass spectrometric conditions are selected ion monitoring; electron impact ionisation at 70 eV; a transfer line temperature of 250 °C; an ion source temperature of 200 °C; a solvent delay of 6 min; and an acquisition window of 6 min to 12 min. Table 4 gives the qualifier ions as m/z 106, 133 and 152 and the quantifier ion as m/z 150 for acrylamide, and the qualifier ions as m/z 108, 136 and 153 and the quantifier ion as m/z 155 for the carbon-13 labelled internal standard.

12.6 The calibration curve is drawn by injecting the derivatised working standard solutions and plotting the ratio of the peak areas of acrylamide and of its internal standard, taken from the quantifier ion mass chromatograms, against the acrylamide concentration. The full scan spectra of the brominated derivatives are given as Figure A.2.

12.7 For qualitative determination the retention time of the analyte peak in the sample is to agree with that in the corresponding standard solution to within plus or minus 0.5%, both the quantifier and the qualifier ions are to appear, and the relative abundance ratio is to agree with that of a standard solution of comparable concentration within the deviations of Table 5, namely plus or minus 10% where the relative abundance is greater than 50%, plus or minus 15% where it is greater than 20% and not greater than 50%, plus or minus 20% where it is greater than 10% and not greater than 20%, and plus or minus 50% where it is not greater than 10%.

12.8 For quantitative determination the derivatised sample solution is injected, the ratio of the peak areas of acrylamide and of the internal standard is obtained and the concentration is read from the calibration curve, the response being kept within the range of the curve. The mass chromatograms of the brominated derivatives are given as Figure A.3.

12.9 A blank test is carried out as in 5.8.

13 Expression of results of the second method

13 The results are expressed as in Clause 6.

14 Precision of the second method

14 The absolute difference between two independent results obtained under repeatability conditions is not to exceed 20% of their arithmetic mean.

15 Other

15 For a test portion of 1 g the detection limit of the method is 3 micrograms per kilogram and the quantification limit 10 micrograms per kilogram.

16 Safety warning

16 The bromine-containing reagent is toxic; gloves and a mask are to be worn and protective measures taken when it is used.