GB 5009.209-2016 PDF in English
GB 5009.209-2016 (GB5009.209-2016) PDF English
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National food safety standard -- Determination of zearalenone in cereals
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| GB/T 5009.209-2008 | English | 239 |
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Determination of zearalenone in cereals
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Standards related to (historical): GB 5009.209-2016
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GB 5009.209-2016: PDF in English GB 5009.209-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Zearalenone in Cereals
ISSUED ON: DECEMBER 23, 2016
IMPLEMENTED ON: JUNE 23, 2017
Issued by: National Health and Family Planning Commission of the
People’s Republic of China;
China 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 ... 9
7 Precision ... 9
8 Others ... 9
9 Principle ... 10
10 Reagents and materials ... 10
11 Instruments and apparatuses ... 11
12 Analysis steps ... 11
13 Description of the analysis result ... 12
14 Precision ... 13
15 Others ... 13
16 Principle ... 13
17 Reagents and materials ... 13
18 Instruments and apparatuses ... 15
19 Analysis steps ... 15
20 Description of the analysis result ... 19
21 Precision ... 19
22 Others ... 19
Appendix A Chromatogram of zearalenone standard ... 20
National Food Safety Standard -
Determination of Zearalenone in Cereals
1 Scope
This Standard specifies methods for the determination of zearalenone in foods.
The method 1 of this Standard is applicable to the determination of zearalenone
in food and food products, alcohol, soy sauce, vinegar, sauce and sauce
products, soybean, rapeseed and edible vegetable oil; method 2 is applicable
to the determination of zearalenone in soybean, rapeseed and edible vegetable
oil; method 3 is applicable to the determination of zearalenone in beef, pork,
beef liver, milk and egg.
Method 1 -- Liquid Chromatography
2 Principle
Use acetonitrile solution to extract zearalenone in the sample; after it is purified
by the immunoaffinity column, use high-performance liquid chromatography
fluorescence detector to measure; use external standard method to quantify.
3 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical
reagents, the water is grade-1 water specified by GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographic pure.
3.1.2 Acetonitrile (CH3CH): chromatographic pure.
3.1.3 Sodium chloride (NaCl).
3.1.4 Potassium chloride (KCl).
3.1.5 Disodium hydrogen phosphate (Na2HPO4).
3.1.6 Monopotassium phosphate (KH2PO4).
glass-fiber filter paper to filter it until the filtrate is clarified; reserve the filtrate
for later use.
5.1.4 Alcohol
Take 20.0 g of degassed alcohol sample (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 acetonitrile to fix-volume to the scale;
shake well. Pipette 10.0 mL of the filtrate; add 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.
5.2 Purification
5.2.1 Food and food products
Connect the immunoaffinity column to a glass syringe; accurately transfer 10
mL (equivalent to 0.8 g of samples) of the filtrate in 5.1; inject into the glass
syringe. Connect the air pressure pump to the glass syringe; adjust the pressure,
so that the solution passes through the immunoaffinity column at a flow rate of
1 drop/s ~ 2 drops/s, until part of the air enters the affinity column. Use 5 mL of
water to rinse the column for one time, at a flow rate of 1 drop/s ~ 2 drops/s,
until part of the air enters the affinity column; discard all the effluent. Accurately
add 1.5 mL of methanol to elute at a flow rate of about 1 drop/s. Collect the
eluate in a glass test tube; use nitrogen to dry at 55°C or lower; then, use 1.0
mL of the mobile phase to dissolve the residue, which is for liquid
chromatography determination.
5.2.2 Soy sauce, vinegar, sauce and sauce products, alcohol
Connect the immunoaffinity column to a glass syringe; accurately transfer 10.0
mL of the filtrate in 5.1.2 or 5.1.4; inject into the glass syringe. Connect the air
pressure pump to the glass syringe; adjust the pressure, so that the solution
slowly passes through the immunoaffinity column at a flow rate of 1 drop/s ~ 2
drops/s, until part of the air enters the affinity column. Successively use 10 mL
of PBS washing buffer and 10 mL of water to rinse the immunoaffinity column,
at a flow rate of 1 drop/s ~ 2 drops/s, until the air enters the affinity column;
discard all the effluent. Accurately add 1.0 mL of methanol to elute at a flow rate
of about 1 drop/s. Collect the eluate in a glass test tube; use nitrogen to dry at
55°C or lower; then, use 1.0 mL of the mobile phase to dissolve the residue,
which is for liquid chromatography determination.
5.2.3 Soybean, rapeseed, edible vegetable oil
Connect the immunoaffinity column to a glass syringe; accurately transfer 10
mL of filtrate in 5.1.3; inject into the glass syringe. Connect the air pressure
Method 2 -- Fluorescence spectrophotometry
9 Principle
Use acetonitrile solution to extract zearalenone in the sample; purify through
the immunoaffinity column; then, add aluminum chloride solution for derivation.
Use a fluorometer to determine the eluent.
10 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical
reagents, the water is grade-1 water specified by GB/T 6682.
10.1 Reagents
10.1.1 Acetonitrile (CH3CH): chromatographic pure.
10.1.2 Methanol (CH3OH): chromatographic pure.
10.1.3 Sodium chloride (NaCl).
10.1.4 Disodium hydrogen phosphate (Na2HPO4).
10.1.5 Monopotassium phosphate (KH2PO4).
10.1.6 Potassium chloride (KCl).
10.1.7 Tween-20 (C58H114O26).
10.1.8 Hydrochloric acid (HCl).
10.1.9 Sulfuric acid (H2SO4).
10.1.10 Aluminum chloride (AlCl3·10H2O).
10.1.11 Quinine sulfate (C20H24N2O2·H2SO4·2H2O).
10.2 Preparation of reagents
10.2.1 Extract: acetonitrile-water (9+1).
10.2.2 PBS/ Tween-20 buffer: weigh 8.0 g of sodium chloride, 1.2 g of disodium
hydrogen phosphate, 0.2 g of monopotassium phosphate, 0.2 g of potassium
chloride in 900 mL of water; use hydrochloric acid to adjust the pH to 7.0; add
1 mL of Tween-20; use water to dilute to 1 L.
12.2 Purification
Connect the immunoaffinity column to a glass syringe; accurately transfer 10
mL of filtrate; inject into the glass syringe. Connect the air pressure pump to the
glass syringe; adjust the pressure, so that the solution slowly passes through
the immunoaffinity column at a flow rate of 1 drop/s ~ 2 drops/s, until part of the
air enters the affinity column. Successively use 10 mL of PBS/ Tween-20 buffer
solution and 10 mL of water to rinse the immunoaffinity column, at a flow rate
of 1 drop/s ~ 2 drops/s, until the air enters the affinity column; discard all the
effluent. Accurately add 1.0 mL of methanol to elute at a flow rate of about 1
drop/s. Collect the eluate in a glass test tube; use nitrogen to dry at 55°C or
lower; then, use 1.0 mL of the mobile phase to dissolve the residue, which is
for determination.
12.3 Blank test
Do not weigh the sample; do a blank test according to the steps of 12.1 and
12.2. It shall be confirmed that it does not contain any substance that interferes
with the to-be-test component.
12.4 Determination
12.4.1 Determination conditions
Excitation wavelength of 360 nm; emission wavelength of 450 nm.
12.4.2 Fluorescence photometer calibration
Use the 0.05 mol/L sulfuric acid solution as a blank for zero setting; use the
fluorescence photometer calibration solution for calibration.
12.4.3 Sample solution determination
Add 1.0 mL of aluminum chloride-derived solution to the sample solution that is
obtained in 12.2; immediately place it in a fluorescence photometer to read the
concentration of zearalenone.
13 Description of the analysis result
Calculate the content of zearalenone in the sample according to Formula (2):
Where:
17.1 Reagents
17.1.1 Methanol (CH3OH): chromatographic pure.
17.1.2 Acetonitrile (CH3CH): chromatographic pure.
17.1.3 Absolute diethyl ether (C4H10O).
17.1.4 Trichloromethane (CHCl3).
17.1.5 Sodium hydroxide (NaOH).
17.1.6 Sodium acetate trihydrate (CH3COONa·3H2O).
17.1.7 Phosphoric acid (H3PO4): purity greater than 85%.
17.1.8 Glacial acetic acid (CH3COOH): chromatographic pure.
17.1.9 β-glucuronide.
17.1.10 Sulfatase.
17.2 Preparation of reagents
17.2.1 Sodium hydroxide solution (0.5 mol/L): weigh 20 g of sodium hydroxide;
use water to dissolve and fix-volume to 1 L.
17.2.2 Sodium acetate buffer solution (0.05 mol/L): weigh 6.8 g of sodium
acetate trihydrate; use 900 mL of water to dissolve; use glacial acetic acid to
adjust the pH to 4.8; fix-volume to 1 L.
17.2.3 Phosphoric acid-water solution: (1+4).
17.2.4 Methanol-water solution: (1+1).
17.2.5 β-glucuronide/ sulfate complex enzyme: 96 000 U/mL β-glucuronide, 390
U/mL sulfatase.
17.3 Standard
Zearalenone (C18H22O5, CAS No.: 17924-92-4), purity ≥ 98.0%. or the standard
substance that is certified by the state and awarded with the standard
substance certificate.
17.4 Preparation of standard solution
17.4.1 Standard stock solution: accurately weigh an appropriate amount of
standard (accurate to 0.000 1 g); use acetonitrile to dissolve it and prepare it a
Note: During the sample preparation, sample contamination or changes in
residue content shall be prevented.
19.1.1 Muscles and internal organs
Take 500 g of sample; use a tissue masher to thoroughly mash and mix; divide
into two portions equally, which are respectively placed in clean containers as
the sample; seal. Store in the dark below -18°C.
19.1.2 Milk
Take 500 g of sample; fully mix; divide into two portions equally, which are
respectively placed in clean containers as the sample; seal. Store at 0°C ~ 4°C
in the dark.
19.1.3 Egg
Take 500 g of sample; remove the shell; use a tissue masher to thoroughly mix;
divide into two portions equally, which are respectively placed in clean
containers as the sample; seal. Store at 0°C ~ 4°C in the dark.
19.2 Hydrolysis
Weigh 5.0 g of sample (accurate to 0.1g) in a 50 mL stoppered centrifuge tube;
add 10 mL of 0.05 mol/L sodium acetate buffer solution and 0.025 mL of β-
glucuronide/ sulfate complex enzyme; vortex and mix; vibrate at a 37°C water
bath for 12 h.
19.3 Extraction
After hydrolysis, add 15 mL of anhydrous diethyl ether; shake and extract for 5
min; then, centrifuge at 4 000 r/min for 2 min; transfer the supernatant to a
concentrated bottle; then, use 15 mL of anhydrous diethyl ether to repeatedly
extract; combine the supernatant; rotate and concentrate at 40°C or below to
nearly dry. Add 1 mL of trichloromethane to dissolve the residue; sonicate for 2
min to help solubilization; transfer to a 10 mL centrifuge tube; then, use 3 mL of
0.5 mol/L sodium hydroxide solution to rinse the concentrated flask; then,
transfer to the same centrifuge tube; vortex to mix; centrifuge at 4 000 r/min for
2 min; absorb the upper sodium hydroxide solution. Then, use 3 mL of 0.5 mol/L
sodium hydroxide to rinse repeatedly and to extract once; combine the sodium
hydroxide extract; add 1 mL of phosphoric acid-water solution; mix well for later
purification.
19.4 Purification
Before using, successively use 5 mL of methanol and 5 mL of water to pre-rinse
the solid phase extraction column.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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