GB 5009.24-2016National food safety standard - Determination of M Aflatoxins in Foods (English PDF)

3.1 Reagents

3.1.1 Acetonitrile (CH3CN). chromatographic purity.

3.1.2 Methanol (CH3OH). chromatographic purity.

3.1.8 Hydrochloric acid (HCl).

3.1.9 Petroleum ether (CnH2n+2). boiling range. 30 °C ~ 60 °C.

3.2 Reagent Preparation

3.2.1 Ammonium acetate solution (5 mmol/L). weigh-take 0.39 g of ammonium acetate;

dilute it in 1,000 mL of water, then, mix it up.

3.2.2 Acetonitrile - water solution (25 + 75). measure-take 250 mL of acetonitrile; add

it to 750 mL of water, then, mix it up.

3.3 Standards

3.3.1 AFT M1 standard (C17H12O7, CAS. 6795-23-9). purity >= 98%, or nationally certified

standard substance with the standard substance certificate.

3.3.2 AFT M2 standard (C17H14O7, CAS. 6885-57-0). purity >= 98%, or nationally certified

standard substance with the standard substance certificate.

3.3.3 13C17-AFT M1 isotope solution (C17H14O7). 0.5 µg/mL.

3.4 Standard Solution Preparation

3.4.1 Standard stock solution (10 µg/mL). respectively weigh-take 1 mg (accurate to

0.01 mg) of AFT M1 and AFT M2; respectively use acetonitrile to dissolve it, then, dilute

to the constant volume of 100 mL.

3.4.2 Mixed standard stock solution (1.0 µg/mL). respectively and accurately absorb

1.00 mL of 10 µg/mL AFT M1 and AFT M2 standard stock solution, then, place them in

the same 10 mL volumetric flask.

3.4.5 5 ng/mL isotope internal standard working solution 2(13C17-AFT M1). take 100 µL

of AFT M1 isotope internal standard (0.5 µg/mL), then, use acetonitrile to dilute to 10

mL. Preserve it at -20 °C; it shall be used for the determination of solid sample. It shall

remain valid for 3 months.

4.1 Balance. division value. 0.01 g, 0.001 g and 0.00001 g.

4.2 Water bath kettle. temperature controlled at 50 °C ± 2 °C.

4.3 Vortex mixer.

4.8 Nitrogen blowing instrument.

4.9 Liquid chromatography - tandem mass spectrometer. equipped with electrospray

ion source.

4.10 Round sieve. 1 mm ~ 2 mm aperture.

4.11 Glass fiber filter paper. rapid, high-load, particle retention in liquid. 1.6 µm.

4.12 Disposable microporous filter head. equipped with 0.22 µm microporous

membrane (before use, selected filter membrane shall adopt standard solution to

confirm that there is no absorption phenomenon).

5.1 Sample Extraction

5.1.1 Liquid milk and yogurt

Weigh-take 4 g (accurate to 0.001 g) of evenly mixed sample, then, place it in a 50 mL

centrifuge tube. Add 100 µL of 13C17-AFT M1 internal standard solution (5 ng/mL); start

oscillation blending, then, evenly place it for 30 min. Add 10 mL of methanol, then, start

vortex for 3 min. Place it at 4 °C; start centrifugation at 6,000 r/min for 10 min, or, filter

it through glass fiber filter paper. Transfer an appropriate amount of the supernatant or

filtrate to a beaker, then, add 40 mL of water or PBS to dilute it; reserve it for later use.

5.1.3 Cream

Weigh-take 1 g (accurate to 0.001 g) of sample, then, place it in a 50 mL centrifuge

tube. Add 100 µL of 13C17-AFT M1 internal standard solution (5 ng/mL); start oscillation

blending, then, evenly place it for 30 min. Add 8 mL of petroleum ether; wait till cream

dissolves, then, add 9 mL of water and 11 mL of methanol. Start oscillation for 30 min.

Transfer all the liquid to separating funnel. Add 0.3 g of sodium chloride, then,

thoroughly shake and dissolve it. Evenly place it and wait for layering, then, transfer

the lower layer to a round-bottomed flask. Start rotary evaporation, till it is below 10

mL, then, use PBS to dilute it to 30 mL.

5.2 Purification

5.2.1 Preparation of immunoaffinity column

Restore immunoaffinity column, which is preserved at low temperature, to room

temperature.

5.2.2 Purification

After abandoning the liquid in the immunoaffinity column, transfer the above-mentioned

sample solution to a 50 mL syringe;

5.3 Liquid Chromatography Reference Conditions

Liquid chromatography reference conditions are listed below.

5.6 Draw a Standard Curve

Under the liquid chromatography - tandem mass spectrometry analytical conditions in

5.3 and 5.4, inject the standard series of solution for determination from low

concentration to high concentration. Use the peak area ratio - concentration of AFT M1

and AFT M2 chromatographic peak and internal standard chromatographic peak 13C17-

AFT M1 to draw the curve. Obtain standard curve regression equation; its linear

correlation coefficient shall be more than 0.99.

5.8 Blank Test

Do not weigh or take sample; comply with the steps in 5.1 and 5.2 to conduct the blank

test. Make sure that there is no substance that would interfere the component to be

determined.