GB 5009.118-2016National food safety standard - Determination of T-2 toxin in foods (English PDF)

3.1 Reagents

3.1.1 Methanol (CH3OH): chromatographic pure.

3.1.2 Acetonitrile (CH3CH): chromatographic pure.

3.1.3 Toluene (C6H5CH3): chromatographic pure.

3.1.4 4-dimethylaminopyridine.

3.1.5 1-anthroylnitrile,1-AN.

3.2 Preparation of reagents

4.7 Air pressure pump.

4.8 Test sieve: aperture of 1.0 mm.

4.9 Balance: sensitivity of 0.000 1 g and 0.01 g.

4.10 Ultrasonic generator: power > 180 W.

4.11 Glass syringe: 10 mL.

5.1 Extraction

5.1.1 Food and food products

Grind the sample; for hard food or the like, use a high-speed pulverizer to grind well, and use a test sieve to filter. Weigh 25.0 g (accurate to 0.1g) of sieved sample in a volumetric flask; use extract to fix-volume to 100 mL; transfer it to a homogenizing cup;

use a homogenizer to stir and extract at high speed for 2 min; use a quantitative filter paper to filter it. Pipette 10.0 mL of the filtrate into 40 mL of water; dilute and mix; use a glass-fiber filter paper to filter it until the filtrate is clarified; reserve the filtrate for later use.

5.1.2 Soy sauce, vinegar, sauce and sauce products

Weigh 25.0 g (accurate to 0.1 g) of the mixed sample; use methanol to fix-volume to

50.0 mL; ultrasonically extract it for 10 min; use quantitative filter paper to filter. Pipette

10.0 mL of the filtrate into 40 mL of water; dilute and mix; use a glass-fiber filter paper to filter it until the filtrate is clarified; reserve the filtrate for later use.

5.1.3 Alcohol

Take 20.0 g (accurate to 0.1 g) of degassed alcohol samples (alcohols which contain carbon dioxide, before using, shall be placed in a refrigerator at 4°C for 30 min, and filtered or ultrasonically degassed) or other alcohol samples which do not contain carbon dioxide in a 50 mL volumetric flask; use methanol to fix-volume to the scale;

shake well; use quantitative filter paper to filter. Pipette 10.0 mL of the filtrate into 40 mL of water; dilute; use a glass-fiber filter paper to filter it until the filtrate is clarified;

reserve the filtrate for later use.

5.2 Purification and elution

Connect the immunoaffinity column to a glass syringe; accurately transfer and inject

10 mL of filtrate which is extracted in 5.1 into the glass syringe. Connect the air pressure pump to the glass syringe; adjust the pressure, so that the solution slowly

Where:

y -- the peak area ratio of the target substance;

a -- the slope of the regression curve;

x -- the concentration of the target substance;

b -- the intercept of the regression curve.

The response values of the test substance in both the standard working solution and the sample solution shall be within the linear response range of the instrument; if the sample content exceeds the standard curve range, it needs be diluted before measurement.

5.5 Blank test

Do not weigh the sample; do a blank test according to the steps of 5.1, 5.2 and 5.3. It shall be confirmed that it does not contain any substance that interferes with the to- be-test component.

5.6 Result calculation

Calculate the content of T-2 toxin in the sample according to Formula (2):

Where:

X -- the content of T-2 toxin in the sample, in micrograms per kilogram (µg/kg);

rho -- the concentration of T-2 toxin in the sample solution, in nanograms per milliliter (ng/mL);

V -- the constant volume after derivatization, in milliliters (mL);

1 000 -- unit conversion coefficient;

m -- the weighing sample, in grams (g);

f -- dilution ratio.

Note: The calculation result needs to be deducted from the blank value, and retain two significant figures.

9.1.5 Ethyl acetate (C4H8O2).

9.1.6 Dimethylformamide (C3H7NO).

9.1.7 Tetramethylbenzidine (TMB).

9.1.8 Tween-20 (C58H114O26).

9.1.9 30% hydrogen peroxide (30% H2O2).

9.1.10 Sodium carbonate (Na2CO3).

9.1.11 Sodium bicarbonate (NaHCO3).

9.1.12 Monopotassium phosphate (KH2PO4).

9.1.13 Disodium hydrogen phosphate (Na2HPO4·12H2O).

9.1.14 Sodium chloride (NaCl)

9.1.15 Potassium chloride (KCl).

9.1.16 Citric acid (C6H8O7·H2O).

9.1.17 Antibody: a specific monoclonal antibody against T-2 toxin that is produced by hybridoma cell lines.

9.1.18 Antigen: conjugate of T-2 toxin and carrier protein-bovine serum albumin (BSA).

9.1.19 Conjugate of rabbit anti-mouse immunoglobulin and horseradish peroxidase

(enzyme-labeled secondary antibody).

9.2 Preparation of reagents

9.2.1 ELISA buffer system.

9.2.1.1 The coating buffer is carbonate buffer of pH 9.6. Weigh 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate; add water to dilute to 1 000 mL.

9.2.1.2 The washing solution is a phosphate buffer (PBS-T for short) of pH 7.4, which contains 0.05% of Tween-20. The preparation method is as follows: weigh 0.2 g of monopotassium phosphate, 2.9 g of disodium hydrogen phosphate, 8.0 g of sodium chloride, 0.2 g of potassium chloride, 0.5 mL of Tween-20; add water to 1 000 mL.

9.2.1.3 The substrate buffer is a phosphoric acid-citric acid buffer of pH 5.0. The preparation method is: 0.1 mol/L of citric acid, that is, weigh 19.2 g of citric acid; add water to 1 000 mL, which is liquid A; AND, 0.2 mol/L of disodium hydrogen phosphate, that is, weigh 71.7 g of disodium hydrogen phosphate; add water to 1 000 mL, which is liquid B. Take 24.3 mL of liquid A and 25.7 mL of liquid B; add water to 100 mL.

Weigh 20.0 g of sample which is pulverized and passed through a 20-mesh sieve;

place it in a 200 mL stoppered conical flask; add 8 mL of water and 100 mL of trichloromethane-absolute ethanol (4:1, volume ratio); stuff up the stopper; shake for

1 h; filter it through a filter paper; take 25 mL of filtrate in an evaporating dish; place it on a water bath at 90°C to volatilize it. Use 50 mL of petroleum ether, in several times, to dissolve the residue in the evaporating dish; wash it into a 250 mL separatory funnel;

use 20 mL of methanol-water (4:1) to wash it for several times; transfer to the same separatory funnel; shake for 1.5 min; let stand for 15 min; collect the lower methanol- water extract; purify it through the chromatography column.

Pour the eluent which passes the column into the evaporating dish; concentrate it to dryness in the water bath; add 3 mL of ethyl acetate while hot; heat to boiling; dry it;

repeat again; lastly, add 3 mL of ethyl acetate; after it becomes as cool as the room temperature, transfer it to the flask. Use an appropriate amount of ethyl acetate to wash the evaporating dish; incorporate it into a flask; place the flask in a 95°C water bath; after it becomes dry and cool, use PBS of 20% methanol to fix-volume, which is used for later ELISA test.

11.2 Test

11.2.1 Use T-2-BSA (4 µg/mL) to coat the enzyme-labeled plate, with 100 µL per well;

let it stand overnight at 4°C.

11.2.2 Use PBS-T to wash the enzyme-labeled plate 3 times; for each time, after 3 minutes, add standard working solution of different concentrations (to make the standard curve) or mixture of sample extract (to test the toxin content in sample) and antibody solution (1:1, volume ratio; 100 µL per well; the mixture shall be prepared the day before use and placed overnight at 4°C); let stand at 37°C for 1 h.

11.2.3 Wash the enzyme-labeled plate 3 times; for each time, after 3 min, add enzyme- labeled secondary antibody, 100 µL per well, at 37°C for 1.5 h.

11.2.4 After the same washing with the above, add the substrate solution, 100 µL per well, at 37°C for 30 min.

11.2.5 Use 1 mol/L sulfuric acid solution to stop the reaction, 50 µL per well; measure the absorbance at 450 nm.

16.1 Reagents

16.1.1 Methanol (CH3OH).

16.1.2 Petroleum ether (C7H7BrMg).

16.1.3 Trichloromethane (CHCl3).

16.1.4 Absolute ethanol (C2H5OH).

16.1.5 Ethyl acetate (C4H8O2).

16.1.6 Dimethylformamide (C3H7NO).

16.1.7 Tetramethylbenzidine (TMB).

16.1.8 Tween-20 (C58H114O26).

16.1.9 30% hydrogen peroxide (30% H2O2).

16.1.10 Conjugate of anti-T-2 toxin monoclonal antibody and horseradish peroxidase.

16.1.11 Antigen: conjugate of T-2 toxin and carrier protein-bovine serum albumin (T-

2-BSA).

16.2 Preparation of reagents

16.2.1 ELISA buffer system.

16.2.1.1 The coating buffer is carbonate buffer of pH 9.6. Weigh 1.59 g of sodium carbonate (Na2CO3), 2.93 g of sodium bicarbonate (NaHCO3); add water to dilute to 1

000 mL.

16.2.1.2 The washing solution is a phosphate buffer (PBS-T for short) of pH 7.4, which contains 0.05% of Tween-20. The preparation method is as follows: weigh 0.2 g of monopotassium phosphate, 2.9 g of disodium hydrogen phosphate, 8.0 g of sodium chloride, 0.2 g of potassium chloride, 0.5 mL of Tween-20; add water to 1 000 mL.

16.2.1.3 Sample diluent: phosphate buffer (PBS for short) of pH 7.2. The preparation

method is as follows: weigh 0.55 g of sodium dihydrogen phosphate (NaH2PO4·H2O),

17.5 Balance: sensitivity of 0.000 1 g, 0.01 g.

17.6 Homogenizer.

17.7 Pulverizer.

18.1 Extraction

18.1.1 Direct ELISA 1

Weigh 20 g of sample which is pulverized and passed through a 20-mesh sieve; place it in a 200 mL stoppered conical flask; add 8 mL of water and 100 mL of trichloromethane-absolute ethanol (4:1, volume ratio); stuff up the stopper; shake for

1 h; filter it through a filter paper; take 25 mL of filtrate in an evaporating dish; place it on a water bath at 90°C to volatilize it. Use 50 mL of petroleum ether, in several times, to dissolve the residue in the evaporating dish; wash it into a 250 mL separatory funnel;

use 20 mL of methanol-water (4:1, volume ratio) to wash it for several times; transfer to the same separatory funnel; shake for 1.5 min; let stand for 15 min; collect the lower methanol-water extract; purify it through the chromatography column.

Pour the eluent which passes the column into the evaporating dish; concentrate it to dryness in the water bath; add 3 mL of ethyl acetate while hot; heat to boiling; dry it;

repeat again; lastly, add 3 mL of ethyl acetate; after it becomes as cool as the room temperature, transfer it to the flask. Use an appropriate amount of ethyl acetate to wash the evaporating dish; incorporate it into a flask; place the flask in a 95°C water bath; after it becomes dry and cool, use PBS of 20% methanol to fix-volume, which is used for later ELISA test.

18.1.2 Direct ELISA 2

Use the method of quartering to reduce the sample to 1 kg; grind all of them into particles of which the size can pass through the 20-mesh sieve; mix well; evenly divide them into two portions as samples; respectively place them into clean containers and seal. Weigh 25.0 g of the sample (accurate to 0.1 g); add 125 mL of methanol-water (7:3, volume ratio); homogenize for 1 min ~ 2 min; centrifuge at 5 000 r/min for 10 min.

After centrifugation, add 50 µL of the filtrate to 300 µL of the sample dilution; mix. Take

50 µL for enzyme-linked immunoassay; the final dilution factor is 35. For high- concentration samples, if the toxin content is beyond the range of the standard curve, it can be further diluted until the toxin concentration in the sample is within the range of the standard curve.

18.2 Test

18.2.1 Direct ELISA 1

Where:

X -- the content of T-2 toxin in the sample, in micrograms per kilogram (µg/kg);

m1 -- the amount of T-2 toxin which is measured on the plate and obtained according to the standard curve, in nanograms (ng);

V1 -- the volume of the sample extract, in milliliter (mL);

V2 -- the volume of the sample solution, in milliliter (mL);

f -- the total dilution factor of the sample solution;

m -- the weighing sample, in grams (g);

Note: The measurement result retains a significant digit after the decimal point.

19.2 Direct ELISA 2

Calculate the content of T-2 toxin in the sample according to Formula (5):

Where:

X -- the content of T-2 toxin in the sample, in micrograms per kilogram (µg/kg);

rho -- T-2 toxin content in the sample that is obtained from the standard working curve, in nanograms per milliliter (ng/mL);

V -- the final volume of the sample solution, in milliliters (mL);

1 000 -- unit conversion coefficient;

m -- the final sample mass that is represented by the sample solution, in grams (g).

Note: The measurement result retains a significant digit after the decimal point.