GB 5009.302-2025 PDF English
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| GB 5009.302-2025 | English | 95 |
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National food safety standard - Determination of Nonylphenol in Food
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GB 5009.302-2025: National food safety standard - Determination of Nonylphenol in Food---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB5009.302-2025
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Determination of
nonylphenol in food
Issued on: MARCH 16, 2025
Implemented on: SEPTEMBER 16, 2025
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope... 3
2 Principle... 3
3 Reagents and materials... 3
4 Instruments and equipment... 4
5 Sample preparation and storage... 5
6 Analysis steps... 5
7 Expression of analysis results... 8
8 Precision... 8
9 Others... 9
Annex A Multiple reaction monitoring (MRM) diagram of 4-nonylphenol standard and
its internal standard... 10
National food safety standard - Determination of
nonylphenol in food
1 Scope
This Standard specifies the liquid chromatography-tandem mass spectrometry method
for the determination of 4-nonylphenol in food.
This Standard applies to the determination of 4-nonylphenol in food.
2 Principle
4-nonylphenol in the sample is extracted with acetonitrile or acetonitrile solution,
separated by high performance liquid chromatography, detected by tandem mass
spectrometer, and quantified by isotope internal standard method.
3 Reagents and materials
Unless otherwise specified, the reagents used in this method are of chromatographic
purity, and the water is grade 1 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH).
3.1.2 Acetonitrile (CH3CN).
3.1.3 Ammonia water (NH3 · H2O). concentration ≥ 25 %.
3.2 Reagent preparation
3.2.1 70 % acetonitrile solution. measure 700 mL of acetonitrile, dilute to 1000 mL with
water.
3.2.2 0.1 % ammonia solution. pipette 1 mL of ammonia solution, dilute to 1000 mL
with water.
3.3 Standards
3.3.1 4-nonylphenol (C15H24O) standard
4-nonylphenol (C15H24O, CAS No.. 84852-15-3), purity ≥ 99 %, or a standard certified
by the state and awarded a reference standard certificate.
3.3.2 Isotope internal standard (3,6,3-nonylphenol-13C6)
3,6,3-nonylphenol-13C6 standard (13C6C9H24O, CAS No.. 1173020-38-6), purity ≥ 99 %.
3.4 Preparation of standard solutions
3.4.1 4-nonylphenol standard stock solution (100 mg/L). accurately weigh 10 mg of 4-
nonylphenol standard (accurate to 0.00001 g), place in a 10 mL beaker, dissolve in
methanol, transfer to a 100 mL brown volumetric flask, make up to the mark with
methanol, mix well, store at -18 ℃, the shelf life is 6 months.
3.4.2 Isotope internal standard stock solution (100 mg/L). accurately weigh 10 mg of
3,6,3-nonylphenol-13C6 standard (accurate to 0.00001 g), place in a 10 mL beaker,
dissolve in methanol, transfer to a 100 mL brown volumetric flask, make up to the mark
with methanol, mix well, store at -18 ℃, the shelf life is 6 months.
3.4.3 4-nonylphenol standard intermediate solution (1.0 mg/L). accurately transfer 1.0
mL of 4-nonylphenol standard stock solution (100 mg/L) into a 100 mL brown
volumetric flask, make up to the mark with methanol, mix well, store at -18°C, the shelf
life is 3 months.
3.4.4 Isotope internal standard working solution (1.0 mg/L). accurately transfer 1.0 mL
of isotope internal standard stock solution (100 mg/L) into a 100 mL brown volumetric
flask, make up to the mark with methanol, mix well, store at -18°C, the shelf life is 3
months.
3.4.5 4-nonylphenol standard series working solutions. accurately pipette 2.50 mL of
4-nonylphenol standard intermediate solution (1.0 mg/L) into 25 mL brown volumetric
flasks, dilute with methanol and make up to the mark, shake well, then respectively
pipette 100 μL, 250 μL, 500 μL, 1.25 mL, 2.50 mL, 5.00 mL of the above solutions and
125 μL of isotope internal standard intermediate solution (1.0 mg/L), place into 25 mL
brown volumetric flasks, make up to the mark with acetonitrile, and mix well. The mass
concentrations of 4-nonylphenol are 0.40 ng/mL, 1.00 ng/mL, 2.00 ng/mL, 5.00 ng/mL,
10.0 ng/mL, and 20.0 ng/mL respectively, and the mass concentration of isotope
internal standard solution in the standard working solution is 5.0 ng/mL. Prepare before
use.
3.5 Materials
3.5.1 Polytetrafluoroethylene centrifuge tubes. 5 mL and 10 mL.
4 Instruments and equipment
4.1 Liquid chromatography-tandem mass spectrometer. equipped with electrospray ion
source.
4.2 Analytical balance. the sensitivity is 0.00001 g and 0.01 g respectively.
and 5.0 mL of 70 % acetonitrile solution (5.0 mL of acetonitrile for meat and aquatic
samples) in sequence, vortex for 30 s, seal with a stopper and ultrasonically extract for
30 min, cool to room temperature, centrifuge at 10000 r/min for 5 min at 4 ℃, pipette
all the upper layer solution into a 5 mL volumetric flask, make up to the mark with
acetonitrile, mix well; if the test solution is still turbid, centrifuge once more, take the
supernatant and determine by liquid chromatography-tandem mass spectrometry.
6.1.4 Milk powder and infant formula
Weigh 1 g of sample (accurate to 0.01 g) into a 5 mL glass graduated tube, add 25.0 μL
of isotope internal standard working solution (1.0 mg/L) and about 1.5 mL of hot water
at about 50 ℃ in sequence, vortex for 10 s to fully dissolve, ultrasonically extract for
10 min, add 3.5 mL of acetonitrile, seal with a stopper and shake vigorously for 5 s,
pipette all the upper layer solution and transfer to a 5 mL polytetrafluoroethylene
centrifuge tube, centrifuge at 10000 r/min for 5 min at 4 ℃, pipette all the upper layer
solution and place in a 5 mL volumetric flask, make up to the mark with acetonitrile,
mix well; if the test solution is still turbid, centrifuge once more, take the supernatant
and determine by liquid chromatography-tandem mass spectrometry.
6.2 Instrument reference conditions
6.2.1 Liquid chromatography reference conditions.
a) Chromatographic column. C18 chromatographic column (4.6 mm × 50 mm, 1.8
μm), equipped with pre-column (4.6 mm × 5 mm, 1.8 μm), or columns with
equivalent performance;
b) Mobile phase. 0.1 % ammonia solution + methanol = 10 + 90 (volume ratio);
c) Flow rate. 0.35 mL/min;
d) Column temperature. 30 ℃;
e) Sample injection volume. 5 μL.
6.2.2 Mass spectrometry reference conditions.
a) Ionization mode. electrospray ionization negative ion mode (ESI－);
b) Mass spectrometry scanning mode. multiple reaction monitoring (MRM);
c) Drying gas temperature. 340 ℃ (nitrogen);
d) Drying gas flow rate. 8 L/min (nitrogen);
e) Nebulizer pressure. 40 psi;
f) Capillary voltage. 4000 V;
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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