GB 5009.185-2016 English PDF (GB5009.185-2016)

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GB 5009.185-2016: Determination of patulin in foods
GB 5009.185-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Patulin in Foods
ISSUED ON: DECEMBER 23, 2016
IMPLEMENTED ON: JUNE 23, 2017
Issued by: National Health and Family Planning Commission of the PRC;
State Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and apparatuses ... 6
5 Analysis steps ... 6
6 Description of the analysis result ... 10
7 Precision ... 10
8 Others ... 10
9 Principle ... 11
10 Reagents and materials ... 11
11 Instruments and apparatuses ... 12
12 Analysis steps ... 13
13 Description of the analysis result ... 14
14 Precision ... 14
15 Others ... 14
Appendix A The calibration of the concentration of patulin standard solution ... 15
Appendix B ... 17
Appendix C Chromatogram ... 20
National Food Safety Standard -
Determination of Patulin in Foods
1 Scope
This Standard specifies methods for the determination of patulin in foods.
Method 1 of this Standard is isotope dilution - liquid chromatography tandem mass
spectrometry, which applies the determination of patulin content in fruits and products
of apple and hawthorn, fruit and vegetable juices and alcoholic foods.
Method 2 of this Standard is high performance liquid chromatography, which applies
the determination of patulin content in products of apple, its fruit and vegetable juices
and alcoholic foods.
Method 1 -- Isotope dilution - liquid chromatography -
tandem mass spectrometry
2 Principle
The patulin in the sample (turbid juice, semi-fluid and solid samples are enzymatically
treated by pectinase) is subjected by solvent extraction, purified and concentrated
through patulin solid-phase purification column or mixed anion-exchange column,
ionized by electrospray ionization source, detected by multiple reaction monitoring,
and quantified by internal standard method.
3 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical reagents, the
water is grade-1 water that is specified by GB/T 6682.
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 Instruments and apparatuses
4.1 Liquid chromatography - mass spectrometer: with electrospray ion source.
4.2 Homogenizer.
4.3 High-speed pulverizer.
4.4 Tissue stamping machine.
4.5 Vortex oscillator.
4.6 pH meter: measurement accuracy ± 0.02.
4.7 Balance: the sensitivity is 0.01 g and 0.000 01 g.
4.8 50 mL PVC centrifuge tube with a plug.
4.9 Centrifuge: speed ≥ 6 000 r/min.
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 Analysis steps
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 Instruments and apparatuses
11.1 Liquid chromatograph: equipped with a UV detector.
11.2 Homogenizer.
11.3 High-speed pulverizer.
11.4 Tissue stamping machine.
11.5 Vortex oscillator.
11.6 pH meter: measurement accuracy ± 0.02.
11.7 Balance: the sensitivity is 0.01 g and 0.000 01 g.
11.8 50 mL PVC centrifuge tube with a plug.
11.9 Centrifuge: speed ≥ 6 000 r/mi...