GB 5009.185-2016National food safety standard - Determination of Patulin in Foods (English PDF)

3.1 Reagents

3.1.1 Acetonitrile (CH3CH): chromatographic pure.

3.1.2 Methanol (CH3OH): chromatographic pure.

3.1.3 Acetic acid (CH3COOH): chromatographic pure.

4.10 Patulin solid-phase purification column (hereinafter referred to as purification

column): mixed filler purification column MycosepTM228 or the equivalent.

4.11 Mixed anion exchange column: a solid-phase extraction column that uses N-

Vinyl-2-pyrrolidone-divinylbenzene copolymer matrix-CH2N(CH3)2C4H9+ as the filler, or the equivalent. Before using, respectively use 6 mL of methanol and 6 mL of water to pre-elute and keep the column moist.

4.12 100 mL pear-shaped flask.

4.13 Solid-phase extraction device.

4.14 Rotary evaporator.

4.15 Nitrogen-blowing instrument.

5.1 Sample preparation

5.1.1 Liquid sample (apple juice, hawthorn juice, etc.)

Pour the sample into a homogenizer and mix; take any 100 g (or mL) of the sample for testing.

Alcohol samples need be degassed by ultrasound for 1 h or be stored at low temperature of 4°C overnight.

residue; use a 0.22 µm filter to filter it; collect the filtrate in the sample bottle for sample injection. Use the same operation method to do a blank test.

5.2.2 Purification column method

5.2.2.1 Sample extraction

5.2.2.1.1 Liquid sample

Weigh 4 g of sample (accurate to 0.01 g) to a 50 mL centrifuge tube; add 250 µL of isotope internal standard working solution; add 21 mL of acetonitrile; mix well;

centrifuge at 6 000 r/min for 5 min; place aside for purification.

5.2.2.1.2 Solid, semi-fluid sample

Weigh 1 g of sample (accurate to 0.01g) in a 50 mL centrifuge tube; add 100 µL of isotope internal standard working solution; mix well and let stand for a while; then, add

10 mL of water and 150 µL of pectinase solution to mix; leave it at room temperature overnight; add 10.0 mL of ethyl acetate; eddy and mix for 5 min; centrifuge at 6 000 r/min for 5 min; transfer the ethyl acetate layer to a pear-shaped flask. Then, use 10.0 mL of ethyl acetate to extract for one more time; combine the two ethyl acetate extracts;

use a rotary evaporator to concentrate it to dryness in the water bath at 40°C; use 2.0 mL of acetic acid solution to dissolve the residue; add 8 mL of acetonitrile; mix well for later purification.

5.2.2.2 Purification

Operate according to the instruction of the purification column which is used; use a purification column to purify the extract; discard the first 1 mL of the purification solution, and collect the subsequent portion.

Use a centrifuge tube to accurately absorb 5.0 mL of purification solution; add 20 µL of acetic acid; use nitrogen to slowly blow it to near dryness at 40°C; use acetic acid solution to fix-volume to 1 mL; roll for 30 s to dissolve the residue; use a 0.22 µm filter to filter it; collect the filtrate in the sample bottle for sample injection. Use the same operation method to do a blank test.

Note: The sample extraction and purification part which is mention above includes mixed anion exchange column purification and purification column purification method, which can be selected according to the actual situation.

5.3 Apparatus reference conditions

5.3.1 Chromatography reference conditions

a) Chromatographic column: T3 chromatographic column with a column length of 100 mm, an inner diameter of 2.1 mm, a particle size of 1.8 µm, or columns of equivalent

10.4 Preparation of standard solution

10.4.1 Standard stock solution (100 µg/mL): use 2 mL of acetonitrile to dissolve 1.0 mg of patulin standard; then, transfer it to a 10 mL volumetric flask; use acetonitrile to fix-volume to the scale. Transfer the solution to a reagent bottle; freeze and store it at

-20°C for use. It is valid for 6 months. See Appendix A for the calibration of the concentration of patulin standard solution.

10.4.2 Standard working solution (1 µg/mL): transfer 100 µL of the calibrated patulin

standard stock solution; use acetic acid solution to dissolve and transfer it to a 10 mL volumetric flask; fix-volume to the scale. Transfer the solution to a reagent bottle; store it at 4°C in the dark for use. It is valid for 3 months.

10.4.3 Standard series working solution: accurately transfer the standard working

solution to the 5 mL volumetric flasks respectively; use acetic acid solution to fix- volume to the mark, so as to prepare the series standard solution with the concentration of patulin of 5 ng/ mL, 10 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL, 150 ng/mL, 200 ng/mL and 250 ng/mL.

11.10 Patulin solid-phase purification column: mixed filler purification column

MycosepTM228 or the equivalent.

11.11 100 mL pear-shaped flask.

11.12 Solid-phase extraction device.

11.13 Rotary evaporator.

Appendix A

The calibration of the concentration of patulin standard solution

A.1 Instrument calibration

Determine the molar extinction coefficient of the potassium dichromate solution, so as to obtain the correction factor of the instrument which is used. Accurately weigh 74 mg of dried potassium dichromate; use sulfuric acid of 0.009 mol/L to dissolve and accurately dilute to 1 000 mL, which is equivalent to [c(K2Cr2O7) = 0.25mmol/L]. Then, absorb 25 mL of this diluent to a 50 mL volumetric flask; add sulfuric acid of 0.009 mol/L to dilute to the mark, which is equivalent to a solution of 0.125 mmol/L. Then, absorb 25 mL of this diluent to a 50 mL volumetric flask; add sulfuric acid of 0.009 mol/L to dilute to the mark, which is equivalent to a solution of 0.062 5 mmol/L. Use a

1 cm quartz cup; use sulfuric acid of 0.009 mol/L as a blank, at the wavelength of the maximum absorption peak (250 nm), to measure the absorbance of the above solutions of three different concentrations. For the molar extinction coefficient of the above three concentrations, calculate according to Formula (A.1):

Where:

E -- the molar extinction coefficient of the potassium dichromate solution;

A -- the measured absorbance of the potassium dichromate solution;

c -- the molar concentration of the potassium dichromate solution.

Compare the average value E' of the molar extinction coefficients of three concentrations with the molar extinction coefficient value 3 160 of potassium dichromate, so as to gain the correction factor of the instrument which is used;

calculate according to Formula (A.2):

Where:

f -- the correction factor of the instrument that is used;

E’ -- the measured potassium dichromate molar extinction coefficient average.

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