GB 5009.111-2016National food safety standard - Determination of deoxynivalenol and its acetylated derivatives in food (English PDF)

1 Scope

The document gives methods for determining deoxynivalenol and its acetylated derivatives in food.

The first method, isotope dilution liquid chromatography-tandem mass spectrometry, applies to the determination of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in cereals and cereal products, alcoholic drinks, soy sauce, vinegar, and pastes and paste products.

The second method, immunoaffinity chromatographic clean-up with high performance liquid chromatography, applies to the determination of deoxynivalenol in the same food groups.

The third method is thin-layer chromatographic determination and the fourth is enzyme-linked immunosorbent screening; both apply to the determination of deoxynivalenol in cereals and cereal products.

5 Analytical procedure, first method

5.1 Sample preparation. For cereals and cereal products at least 1 kg is taken, ground in a high speed mill and sieved so that the particle size passes a test sieve of 0.5 mm to 1 mm aperture, then mixed and reduced to 100 g and kept sealed. For alcoholic drinks at least 1 L of bulk product, or at least three packages of the same batch, is homogenised in one vessel and reduced to 100 g or mL; carbonated samples are refrigerated at 4 °C for 30 min and filtered or degassed by ultrasound before use. Soy sauce, vinegar, pastes and paste products are treated in the same way starting from at least 1 L.

5.2 Extraction. For cereals and cereal products 2 g are weighed to 0.01 g into a 50 mL centrifuge tube, 400 µL of the mixed isotope internal standard working solution is added, the tube is shaken and left 30 min, 20.0 mL of acetonitrile-water (84 + 16) is added and the tube is sonicated or shaken 20 min, then centrifuged 5 min at 10 000 r/min; the supernatant is kept as supernatant A. For alcoholic drinks 5 g are weighed, 200 µL of internal standard is added, the tube is left 30 min, made to 10 mL with acetonitrile and treated in the same way to give supernatant B. Soy sauce, vinegar, pastes and paste products follow the cereal procedure with 2 g and 400 µL and give supernatant C.

5.3 Clean-up. One of three routes is chosen. On the general purpose solid phase extraction cartridge, 5 mL of supernatant is mixed with 10 mL of acetonitrile-saturated hexane, vortexed 2 min and centrifuged 2 min at 5 000 r/min, the hexane layer is discarded, the residue is blown to dryness with nitrogen at 40 °C to 50 °C and taken up in 4 mL of water; the cartridge is conditioned with 3 mL of methanol and 3 mL of water, the sample is loaded at one to two drops per second, the cartridge is washed with 3 mL of water and 1 mL of 5 % methanol in water and dried, and the analytes are eluted with 4 mL of methanol, blown dry, redissolved in 1.0 mL of initial mobile phase and filtered through a 0.22 µm membrane. On the dedicated cartridge, 8 mL of supernatant is placed in the glass tube, the packing tube is pushed in slowly until purified liquid appears, and 5 mL of it is blown dry and taken up as before. On the immunoaffinity column, 5 mL of supernatant is blown dry, taken up in 2 mL of water, passed through the column at one drop per second with an air pressure pump, washed with 5 mL of phosphate buffered saline and 5 mL of water, eluted with 2 mL of methanol at one drop per second, blown almost dry at 50 °C, taken up in 1.0 mL of initial mobile phase by vortexing 30 s and filtered.

5.4 Reference chromatographic and mass spectrometric conditions are given for a positive electrospray source and for a negative electrospray source. Both use a C18 column 100 mm long with an internal diameter of 2.1 mm and 1.7 µm packing, a flow rate of 0.35 mL/min, a column temperature of 40 °C, an injection volume of 10 µL, a desolvation gas flow of 900 L/h and the same gradient, 2 % B from 0 min to 0.8 min, 24 % B from 3.0 min to 4.0 min, 100 % B from 6.0 min to 6.9 min and 2 % B from 6.9 min to 7.0 min. In positive mode the mobile phases are 0.1 % formic acid and 0.1 % formic acid in acetonitrile, the capillary voltage is 3.5 kV, the cone voltage 30 V and the desolvation temperature 350 °C. In negative mode they are 0.01 % ammonia and acetonitrile, the capillary voltage is 2.5 kV, the cone voltage 45 V and the desolvation temperature 500 °C. Tables 1 and 2 list, for each compound, the precursor ion, the cone voltage, the quantifier ion and its collision voltage and the qualifier ion and its collision voltage; a note states that because 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol are isomers, the carbon-13 labelled 3-acetyl compound may be used as the internal standard for the 15-acetyl compound.

5.5 Identification. The retention time of the target peak in the sample varies from that of the corresponding standard peak by not more than +/- 2.5 %. The qualifying ions appear, at least one precursor and two product ions, and within one analytical batch the relative abundance ratio of the two product ions differs from that of a standard solution of comparable concentration by not more than the deviation given in Table 3, which allows +/- 20 % where the relative abundance is above 50 %, +/- 25 % from 20 % to 50 %, +/- 30 % from 10 % to 20 % and +/- 50 % at or below 10 %.

5.6 to 5.8 The calibration curve is drawn from the ratio of the peak area of each compound to that of its internal standard against concentration, and the linear correlation coefficient is greater than 0.99. The sample response falls inside the linear range, otherwise the test portion is reduced and the determination repeated. A blank test is run without the sample.

6 The content is calculated with formula (1). The formula as printed in the source is broken and is not reproduced here. Its symbols are the content of deoxynivalenol, 3-acetyl-deoxynivalenol or 15-acetyl-deoxynivalenol in the sample in micrograms per kilogram; the mass concentration read from the calibration curve by the internal standard method, in nanograms per millilitre; the volume of the sample extract, the final volume of the sample, and the aliquot volume taken for clean-up, all in millilitres; the conversion factor 1000; and the test portion in grams. Results are given to three significant figures.

7 The absolute difference between two independent determinations obtained under repeatability conditions does not exceed 23 % of their arithmetic mean.

8 With a 2 g test portion of cereals and cereal products, alcoholic drinks, soy sauce, vinegar, pastes and paste products, the limits of detection for deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol are 10 µg/kg and the limits of quantification 20 µg/kg. With a 5 g test portion of alcoholic drinks the limits of detection are 5 µg/kg and the limits of quantification 10 µg/kg.

12 Analytical procedure, second method

12.1 Sample preparation is as in 5.1.

12.2 Extraction. For cereals and cereal products 25 g of ground sample are weighed to 0.1 g into a 100 mL stoppered conical flask with 5 g of polyethylene glycol and 100 mL of water, mixed and sonicated or shaken 20 min, then filtered through glass fibre paper until the filtrate is clear, or centrifuged 10 min at 6 000 r/min, and centrifuged 5 min at 10 000 r/min to give filtrate A. For alcoholic drinks 20 g of sample are taken to 0.1 g with 1 g of polyethylene glycol, made to 25.0 mL with water and treated in the same way to give filtrate B. For soy sauce, vinegar, pastes and paste products 25 g are taken to 0.1 g with 5 g of polyethylene glycol, made to 100 mL with water and treated in the same way to give filtrate C.

12.3 Clean-up. The immunoaffinity column stored cold is brought to room temperature and allowed to drain. 2.0 mL of filtrate A, B or C is transferred to a glass syringe barrel connected to an air pressure pump and passed through the column at one drop per second until air enters the column. The column is washed with 5 mL of phosphate buffered saline and 5 mL of water at one to two drops per second until air enters it, all effluent is discarded and the column is drawn dry.

12.4 Elution. Exactly 2 mL of methanol is added and the column eluted at one drop per second, the whole eluate is collected in a tube and blown gently almost to dryness with nitrogen at 50 °C, 1.0 mL of initial mobile phase is added, the residue is dissolved by vortexing 30 s, and the solution is filtered through a 0.45 µm membrane into a sample vial.

12.5 Reference liquid chromatographic conditions: C18 column 150 mm long with an internal diameter of 4.6 mm and 5 µm packing, mobile phase methanol plus water (20 + 80), flow rate 0.8 mL/min, column temperature 35 °C, injection volume 50 µL, detection wavelength 218 nm.

12.6 and 12.7 The calibration curve is drawn with the concentration of the deoxynivalenol working standards on the horizontal axis and the integrated peak area on the vertical axis, the series being injected from low to high. Sample responses fall inside the linear range, otherwise the test portion is reduced and the sample reprocessed. A blank test is run without the sample.

13 The content is calculated with formula (2), which as printed is broken and is not reproduced here. Its symbols are the content of deoxynivalenol in micrograms per kilogram, the mass concentration of deoxynivalenol in the sample and in the blank in nanograms per millilitre, the final volume of the eluate in millilitres, the dilution factor of the sample solution, the conversion factor 1000, and the test portion in grams. Results are given to three significant figures.

14 The absolute difference between two independent determinations under repeatability conditions does not exceed 23 % of their arithmetic mean.

15 With a 25 g test portion of cereals and cereal products, soy sauce, vinegar, pastes and paste products, the limit of detection for deoxynivalenol is 100 µg/kg and the limit of quantification 200 µg/kg; with a 20 g test portion of alcoholic drinks they are 50 µg/kg and 100 µg/kg.

19 Analytical procedure, third method

19.1 Extraction. 20 g of ground sample are weighed into a 200 mL stoppered conical flask. The source text at this point reads that 8 mL and 100 mL of chloroform-absolute ethanol (8 + 2) are added; the reagent that goes with the 8 mL is missing from the document and is not supplied here. The flask is stoppered, a layer of water is put on the stopper to seal it, the flask is shaken 1 h, the contents are filtered through folded rapid qualitative paper, and 25 mL of the filtrate is taken into a 75 mL glass evaporating dish and evaporated to dryness on a 90 °C water bath under ventilation.

19.2 Clean-up. In the liquid-liquid partition step the residue of a cereal sample is dissolved in portions with 50 mL of petroleum ether and washed into a 100 mL separating funnel, the dish being rinsed with 20 mL of methanol-water (4 + 1), 30 mL for maize samples, into the same funnel; for cereal products such as cake, biscuits and bread 100 mL of petroleum ether and a 250 mL funnel are used with 30 mL of methanol-water (4 + 1). The funnel is shaken 1.5 min and left about 15 min to separate, and the lower methanol-water layer is passed to the column, the white flocculent material at the interface being left behind. In the column clean-up step about 0.1 g of degreased cotton is packed tightly at the lower joint of the column, 0.5 g of neutral alumina is added and levelled and 0.4 g of activated carbon is added and tapped down; the column is fitted through a rubber stopper into a filter flask holding a flat-bottomed tube, gentle suction is applied to compact the carbon, the methanol-water extract is added carefully down the wall and drawn through at 18 to 20 drops per 15 s, that is 3 mL/min, and near the end 10 mL of methanol-water (4 + 1) is added to wash the column, which is drawn until no more liquid runs out. The overall rate through the column is held between 2 mL/min and 3 mL/min, since too fast a rate cleans poorly and too slow a rate wastes time. For maize the amount of activated carbon is 0.3 g, the rest being as for wheat and its products.

19.3 Preparation of the solution for thin-layer chromatography. The eluate is poured into a 75 mL evaporating dish, the flat-bottomed tube being rinsed with a little methanol-water (4 + 1), and the dish is concentrated to dryness on a boiling water bath. For wheat, 3 mL of ethyl acetate is added while hot, heated to boiling and the dish swung gently several times so that the analyte is taken up, the solvent is evaporated, the treatment is repeated with a further 3 mL, and a final 3 mL is heated to boiling, cooled to room temperature and transferred to the concentration flask, the dish being rinsed with three 1.5 mL portions of ethyl acetate. For wheat products and maize the first ethyl acetate treatment is followed by about 0.5 mL of methanol-acetone (1 + 2) used to dissolve the residue with a glass rod, evaporation of that solvent, and a further 3 mL of ethyl acetate brought to the boil; the sequence is repeated once. The combined extract is concentrated to dryness in the flask on a water bath at about 95 °C under nitrogen, cooled and taken up in 0.2 mL of chloroform-acetonitrile (4 + 1).

19.4 Determination. Plates are made from 4 g of silica gel G with about 9 mL of 15 % aluminium chloride solution, ground about 2 min to a paste, spread as three plates 5 cm by 20 cm, dried at room temperature, activated 1 h at 105 °C and kept in a desiccator. Spotting is on a baseline 2.5 cm from the lower edge. On the first plate 25 µL of sample solution is spotted 1.8 cm from the left edge and 2 µL of deoxynivalenol standard, 50 ng, is spotted on a line 1.5 cm from the top of the plate opposite the sample spot. Where the first plate shows no fluorescence, a second plate is spotted 0.8 cm to 1 cm from the left edge with an estimated or diluted amount of sample solution, with two standard spots 2 cm from the left edge and 1.2 cm from the right edge that may be 50 ng, 75 ng or 100 ng, and with three standard spots of 50 ng each on the line 1.5 cm from the top corresponding to the three baseline spots. Cross development uses diethyl ether, diethyl ether-acetone (95 + 5) or anhydrous diethyl ether, one of them chosen so that the deoxynivalenol spot moves 0.7 cm to 1 cm off the origin and just separates from the interfering fluorescence; 10 mL is poured into the tank, the plate is dipped along the long edge nearest the spots, developed to the end of the plate for 1 min to 2 min and dried in air for 3 min, and for wheat products a second cross development with 10 mL of petroleum ether boiling between 30 °C and 60 °C is run to the end of the plate for a further 1 min followed by 5 min of drying. Vertical development uses chloroform-acetone-isopropanol (8 + 1 + 1) or chloroform-acetone-isopropanol-water (7.5 + 1 + 1.5 + 0.1); 10 mL is poured into the tank, the plate is developed 15 cm and dried in air 10 min, and where separation is poor the four listed development modes are tried in order of increasing polarity, two of them with water-saturated filter paper lining the tank lid or the tank.

19.4.4 and 19.4.5 Before heating, interfering spots fluoresce blue-violet while deoxynivalenol does not; after the plate is heated 7 min to 10 min at 130 °C in an oven, cooled on a cold surface for 1 min to 5 min and viewed under 365 nm ultraviolet light, deoxynivalenol fluoresces. The three standard spots left undeveloped at the top of the plate serve as lateral position markers for the developed sample and standard spots, so that the sample spot is located both across and along the plate, and its retardation factor is compared with that of the developed standards. Where the first plate shows no fluorescent spot and, on the second plate, the fluorescence of 25 µL of sample solution spiked with 25 ng of standard equals that of 25 ng of standard, the deoxynivalenol content is negative or below 50 µg/kg.

19.4.6 For densitometric measurement the excitation wavelength is 340 nm and the emission wavelength 400 nm. The response is linear with the amount of deoxynivalenol only where the standard spots are at least at 100 ng, 200 ng and 400 ng, and the densitometer is used only for samples containing more than 300 µg/kg; at 300 µg/kg a 20 µL spot brings the measured amount between 100 ng and 200 ng. Two standard spots are put on each plate, of 100 ng and 200 ng or of 200 ng and 400 ng, and the calibration curve is drawn from the measured peak areas against the amount of deoxynivalenol.

20 to 22 The content is calculated with formula (3), which as printed is broken and is not reproduced here; its symbols are the content of deoxynivalenol in micrograms per kilogram, the amount measured in the sample spot in nanograms, the volume of chloroform-acetonitrile used to dissolve the residue and the volume of sample solution spotted, both in millilitres, the total dilution factor, the conversion factor 1000, and the mass of sample corresponding to the dissolved residue in grams. The result is expressed to the units place. Two portions of each sample are analysed in parallel and the arithmetic mean is reported, the relative difference between them being not more than 60 %. The minimum detectable amount on the plate is 100 ng and the limit of detection 300 µg/kg. Samples above the limit set in GB 2761 are confirmed by the first method.

26 Analytical procedure, fourth method

23 Principle. Deoxynivalenol is extracted from the sample with water and the supernatant is obtained by homogenising, vortexing and centrifuging or filtering. An enzyme-labelled deoxynivalenol conjugate competes with the deoxynivalenol of the sample supernatant or of the standard for the specific antibody pre-coated in the microwell. After washing, the corresponding chromogen is added, the reaction is stopped with a mineral acid and the plate is read at 450 nm or 630 nm. Within a given concentration range the deoxynivalenol content of the sample is inversely proportional to the absorbance.

26.1 Extraction. At least 100 g of sample is ground and passed through a test sieve of 1 mm to 2 mm aperture. 5.0 g of the ground sample is taken into a 50 mL centrifuge tube, the extraction solution required by the kit is added, and the determination follows the method described in the kit instructions.

26.2 The enzyme-linked immunosorbent kit is used for the quantitative determination of the test solution according to the operating steps it prescribes.

27 The standard working curve is drawn from the relationship between standard concentration and absorbance using the calculation method or software supplied with the kit; the absorbance of the test solution is entered in that curve to obtain its concentration, and the content in the food is calculated with formula (4), which as printed is broken and is not reproduced here. Its symbols are the content of deoxynivalenol in the food in micrograms per kilogram, the concentration of deoxynivalenol in the test solution in micrograms per litre, the volume of the extract in litres, the dilution factor applied during sample preparation, and the test portion in kilograms. The result is kept to one decimal place, and samples above the limit set in GB 2761 are confirmed by the first method.

28 and 29 The absolute difference between two independent determinations under repeatability conditions does not exceed 25 % of their arithmetic mean. With a 5 g test portion of cereals and cereal products the limit of detection of the method is 200 µg/kg and the limit of quantification 250 µg/kg.

Annex A Reference substances and verification of the immunoaffinity column

A.1 Table A.1 gives the Chinese name, English name, abbreviation, CAS number, molecular formula and relative molecular mass of the three reference substances. Deoxynivalenol, DON, has CAS number 51481-10-8 and relative molecular mass 296; 3-acetyl-deoxynivalenol, 3-ADON, has CAS number 50722-38-8 and relative molecular mass 338; 15-acetyl-deoxynivalenol, 15-ADON, has CAS number 88337-96-6 and the same relative molecular mass, 338.

A.2.1 Column capacity verification. 6000 ng of deoxynivalenol stock standard solution is added to 5 mL of water and mixed thoroughly. Three immunoaffinity columns of the same batch are taken and 1 mL is loaded on each. After loading, washing and elution, the eluate is collected, blown down with nitrogen and made to 1 mL with initial mobile phase, and the deoxynivalenol content is measured by liquid chromatography. The acceptance sentence as printed reads that a result of 1000 ng or more of deoxynivalenol, with a recovery of 80 % or more and a relative standard deviation of 15 % or more, means the product may be used.

A.2.2 Column recovery verification uses the same loading, washing, elution and measurement, and its acceptance sentence as printed reads that a column recovery of 80 % or more, with a relative standard deviation of 15 % or more, means the product may be used.

Amendment No. 1 Amendment No. 1 to GB 5009.111-2016

The amendment was approved by Announcement No. 2 of 16 March 2025 of the National Health Commission and the State Administration for Market Regulation and takes effect from 16 September 2025.

In clause 1 the field of the first method is widened from cereals and cereal products to cereals and cereal products including filled flour and rice products, baked foods made from wheat flour including filled baked foods, and cereal-based complementary foods for infants and young children, alongside alcoholic drinks, soy sauce, vinegar and pastes and paste products.

In 3.4.4 the mixed standard series is extended by one level, so that it becomes 10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL, 160 ng/mL, 320 ng/mL, 640 ng/mL and 1280 ng/mL.

In 5.1.1 the same widening of the food groups is made, and the grinding instruction changes from grinding and sieving so that the particle size passes a sieve of 0.5 mm to 1 mm aperture to grinding so that the particle size is 0.5 mm to 1 mm.

In 5.2.1 and in clause 8 the food groups are widened in the same terms.