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GB 5009.24-2016

Chinese Standard: 'GB 5009.24-2016'
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Detail Information of GB 5009.24-2016; GB5009.24-2016
Description (Translated English): National food safety standard Determination of aflatoxins M1 and B1 in foods
Sector / Industry: National Standard
Classification of Chinese Standard: X09
Word Count Estimation: 22,277
Date of Issue: 2016-12-23
Date of Implementation: 2017-06-23
Older Standard (superseded by this standard): GB 5009.24-2010 Partial; GB 5413.37-2010; GB/T 23212-2008 Partial; SN/T 1664-2005 Partial
Regulation (derived from): National Health and Family Planning Commission Notice No.17 of 2016
Issuing agency(ies): General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, China National Standardization Administration Committee

GB 5009.24-2016
National Food Safety Standard -- Determination of M Aflatoxins in Foods
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of M Aflatoxins in Foods
食品安全国家标准
食品中黄曲霉毒素M族的测定
Issued on. December 23, 2016 Implemented on. June 23, 2017
Issued by. National Health and Family Planning Commission of the PRC;
China Food and Drug Administration.
Table of Contents
Foreword ...4 
1 Scope ...5 
Method I -- Isotope Dilution Liquid Chromatography - Tandem Mass
Spectrometry ...5 
2 Principle ...5 
3 Reagents and Materials ...5 
4 Instruments and Apparatuses ...7 
5 Analytical Procedure ...8 
6 Expression of Analytical Result ...13 
7 Precision ...13 
8 Others ...13 
Method II -- High-performance Liquid Chromatography ...14 
9 Principle ...14 
10 Reagents and Materials ...14 
11 Instruments and Apparatuses ...15 
12 Analytical Procedure ...16 
13 Expression of Analytical Result ...17 
14 Precision ...18 
15 Others ...18 
Method III -- Enzyme-linked Immunosorbent Assay ...18 
16 Principle ...18 
17 Reagents and Materials ...19 
18 Instruments and Apparatuses ...19 
19 Analytical Procedure ...19 
20 Expression of Analytical Result ...20 
21 Precision ...21 
22 Others ...21 
Appendix A Standard Concentration Calibration Method for AFT M1 and AFT M2
...22 
Appendix B Column Capacity Verification Method for Immunoaffinity Column
...23 
Appendix C Liquid Chromatography - Mass Spectrogram and Sub-ion Scan 24 
Appendix D Liquid Chromatogram ...27 
Appendix E Mass Determination Method of Enzyme-linked Immunosorbent
Assay Kit ...28 
National Food Safety Standard -
Determination of M Aflatoxins in Foods
1 Scope
This Standard specifies the methods for the determination of aflatoxins M1 and
aflatoxins M2 (hereinafter referred to as AFT M1 and AFT M2) in foods.
Method I is isotope dilution liquid chromatography - tandem mass spectrometry, which
is applicable to the determination of AFT M1 and AFT M2 in milk, milk products and milk-
containing special dietary food.
Method II is high-performance liquid chromatography; its scope of application is the
same as Method I.
Method III is enzyme-linked immunosorbent screening assay, which is applicable to
the screening determination of AFT M1 in milk, milk products and milk-containing
special dietary food.
Method I -- Isotope Dilution Liquid Chromatography -
Tandem Mass Spectrometry
2 Principle
Use methanol - water solution to extract aflatoxins M1 and aflatoxins M2 in sample.Use
water or phosphate buffer solution to dilute the supernatant, then, purify and enrich it
through immunoaffinity column.After concentration, constant-volume and filtering of
the purified liquid, separate it through liquid chromatography.Detect it through tandem
mass spectrometry, then, quantify it through isotope internal standard method.
3 Reagents and Materials
Unless it is otherwise stipulated, all reagents adopted in this Method shall be analytical
pure; water shall be Grade-1 water stipulated in GB/T 6682.
3.1 Reagents
3.1.1 Acetonitrile (CH3CN). chromatographic purity.
calibrate the concentration (please refer to Appendix A for the calibration method).
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.Add acetonitrile to dilute to the constant volume, then,
obtain 1.0 μg/mL mixed standard solution.This solution can be preserved in an airtight
container in the dark at 4 °C; it shall remain valid for 3 months.
3.4.3 Mixed standard working solution (100 ng/mL). accurately absorb 1.0 mL of mixed
standard stock solution (1.0 μg/mL), then, place it in a 10 mL volumetric flask.Use
acetonitrile to dilute to the constant volume.This solution can be preserved in an
airtight container in the dark at 4 °C; it shall remain valid for 3 months.
3.4.4 50 ng/mL isotope internal standard working solution 1(13C17-AFT M1). take 1 mL
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 liquid sample.It shall
remain valid for 3 months.
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.
3.4.6 Standard series of working solution. respectively and accurately absorb 5 μL, 10
μL, 50 μL, 100 μL, 200 μL and 500 μL of standard working solution, then, place them
in 10 mL volumetric flask.Add 100 μL of 50 ng/mL isotope internal standard working
solution.Use initial mobile phase to dilute to the constant volume.Thus, prepare AFT
M1 and AFT M2 series of standard solution at the concentration of 0.05 ng/mL, 0.1
ng/mL, 0.5 ng/mL, 1.0 ng/mL, 2.0 ng/mL and 5.0 ng/mL.
4 Instruments and Apparatuses
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.
4.4 Ultrasonic cleaner.
4.5 Centrifuge. ≥ 6,000 r/min.
4.6 Rotary evaporator.
4.7 Solid phase extraction device (equipped with vacuum pump).
4.8 Nitrogen blowing instrument.
vortex mixing.If milk powder cannot completely dissolve, place the centrifuge tube in
50 °C water bath; after the milk powder completely dissolves, take it out.Wait till the
sample solution cools down to 20 °C, then, add 10 mL of methanol; 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.
5.1.4 Cheese
Weigh-take 1 g (accurate to 0.001 g) of already shredded and evenly mixed sample
(through aperture 1 mm ~ 2 mm round sieve), 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 1 mL of water and 18 mL of methanol.
Start oscillation for 30 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 round-bottomed flask.Start rotary evaporation, till it is below
2 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; adjust the flow rate of dropping to 1 mL/min ~ 3
mL/min.Wait till the dropping of the sample solution finishes, add 10 mL of water to
the syringe.At a stable flow rate, rinse the immunoaffinity column.Wait till water
droplets stop, then, use the vacuum pump to drain the immunoaffinity column.Isolate
from the vacuum system; underneath the affinity column, place a 10 mL scale test tube;
take down the 50 mL syringe.Add 2 x 2 mL of acetonitrile (or methanol) to elute the
affinity column.Control the flow rate of dropping at 1 mL/min ~ 3 mL/min.Use the
vacuum pump to drain the affinity column, then, gather all the eluent to the scale test
When weighing-taking 1 g of milk powder, special dietary food, cream and cheese, this
Method’s detection limit of AFT M1 is 0.02 μg/kg; the detection limit of AFT M2 is 0.02
μg/kg; the quantitation limit of AFT M1 is 0.05 μg/kg; the quantitation limit of AFT M2 is
0.05 μg/kg.
Method II -- High-performance Liquid Chromatography
9 Principle
Use methanol - water solution to extract aflatoxins M1 and aflatoxins M2 in sample.
After diluting the supernatant, purify and enrich through the immunoaffinity column.
After concentration, constant-volume and filtering of the purified liquid, separate it
through liquid chromatography.Detect it through fluorescence detector, then, quantify
it through external standard method.
10 Reagents and Materials
Unless it is otherwise stipulated, all reagents adopted in this Method shall be analytical
pure; water shall be Grade-1 water stipulated in GB/T 6682.
10.1 Reagents
10.1.1 Acetonitrile (CH3CN). chromatographic purity.
10.1.2 Methanol (CH3OH). chromatographic purity.
10.1.3 Sodium chloride (NaCl).
10.1.4 Disodium phosphate (Na2HPO4).
10.1.5 Potassium dihydrogen phosphate (KH2PO4).
10.1.6 Potassium chloride (KCl).
10.1.7 Hydrochloric acid (HCl).
10.1.8 Petroleum ether (CnH2n+2). boiling range. 30 °C ~ 60 °C.
10.2 Reagent Preparation
10.2.1 Acetonitrile - water solution (25 + 75). measure-take 250 mL of acetonitrile; add
it to 750 mL of water, then, mix it up.
10.2.2 Acetonitrile - methanol solution (50 + 50). measure-take 500 mL of acetonitrile;
11.3 Vortex mixer.
11.4 Ultrasonic cleaner.
11.5 Centrifuge. rotating speed ≥ 6,000 r/min.
11.6 Rotary evaporator.
11.7 Solid phase extraction device (equipped with vacuum pump).
11.8 Nitrogen blowing instrument.
11.9 Round sieve. 1 mm ~ 2 mm aperture.
11.10 Liquid chromatography (equipped with fluorescence detector).
11.11 Glass fiber filter paper. rapid, high-load, particle retention in liquid. 1.6 μm.
11.12 Disposable microporous filter head. equipped with 0.22 μm microporous filter
membrane.
11.13 Immunoaffinity column. column capacity ≥ 100 ng (please refer to Appendix B
for the method of testing and verifying column capacity, recovery rate and column
recovery rate).
NOTE. before use, the mass of different batches of affinity column needs to be verified.
12 Analytical Procedure
The application of immunoaffinity columns provided by different manufacturers might
be slightly different in sample operation, such as sample loading, leaching and elution.
Thus, operation shall comply with the requirements in the operating instructions
provided by the manufacturers.
Warning. the whole analysis and operation process shall be conducted in an
appointed area.This area shall be kept away from light (direct sunlight);
be equipped with relatively independent operating bench and waste
storage device.During the whole experiment process, operators shall
adopt corresponding protective measures in accordance with the
requirements of exposure to violent toxicity.
12.1 Sample Extraction
Do not add isotope internal standard solution.Other than this, the method is the same
as 5.1.
12.2 Purification
Where,
X---content of AFT M1 or AFT M2 in the sample, expressed in (μg/kg);
---concentration of AFT M1 or AFT M2 in the inject solution in the chromatographic
peak through the standard curve, expressed in (ng/mL);
V---final constant volume of the sample after the immunoaffinity column’s purification
and elution, expressed in (mL);
f---dilution factor of the sample solution;
1,000---conversion factor;
m---weighing mass of the sample, expressed in (g).
The calculation result shall retain 3 significant figures.
14 Precision
The absolute difference of two independent determination results obtained under
repeatability conditions shall not exceed 20% of the arithmetic mean value.
15 Others
When weighing-taking 4 g of liquid milk and yogurt, this Method’s detection limit of AFT
M1 is 0.005 μg/kg; the detection limit of AFT M2 is 0.0025 μg/kg; the quantitation limit
of AFT M1 is 0.015 μg/kg; the quantitation limit of AFT M2 is 0.0075 μg/kg.
When weighing-taking 1 g of milk powder, special dietary food, cream and cheese, this
Method’s detection limit of AFT M1 is 0.02 μg/kg; the detection limit of AFT M2 is 0.01
μg/kg; the quantitation limit of AFT M1 is 0.05 μg/kg; the quantitation limit of AFT M2 is
0.025 μg/kg.
Method III -- Enzyme-linked Immunosorbent Assay
16 Principle
Through certain treatment, such as homogenization, frozen centrifugation, degreasing
Add water to dissolve it, then, transfer to a 100 mL volumetric flask.Use water to dilute
to the constant volume.The following step is the same as 19.1.1.
19.1.3 Cheese
Weigh-take 50 g (accurate to 0.1 g) of test sample, then, remove the inedible part on
the surface.In terms of hard cheese, use a grinder to directly grind it.In terms of soft
cheese, freeze it overnight at -20 °C, then, immediately, use a grinder to grind it.Weigh-
take 5 g (accurate to 0.1 g) of evenly mixed test sample, then, add the extract provided
by the kit.Start extraction in accordance with the instruction provided by the kit; the
extract shall be considered as the test solution.
19.2 Quantitative Detection
In accordance with the operational procedure described by the enzyme-linked
immunosorbent assay kit, conduct quantitative detection of the test sample (solution).
20 Expression of Analytical Result
20.1 Draw a Standard Working Curve of Enzyme-linked Immunosorbent
Assay Kit
In accordance with the standard substance’s concentration and absorbance variation
relation, draw a standard working curve.
20.2 Concentration Calculation of Test Solution
Substitute the absorbance of the test solution into the formula obtained in 20.1, then,
calculate the concentration  of the test solution.
20.3 Result Calculation
The content of AFT M1 in foods shall be calculated in accordance with Formula (3).
Where,
X---content of AFT M1 in foods, expressed in (μg/kg);
---concentration of AFT M1 in the test solution, expressed in (μg/L);
V---constant volume (in terms of milk powder, special dietary food and liquid sample)
or extract volume (in terms of cheese) expressed in (L);
f---dilution factor;
Appendix B
Column Capacity Verification Method for Immunoaffinity Column
B.1 Column Capacity Verification
In 30 mL of PBS, add 300 ng of AFT M1 standard stock solution; thoroughly mix it up.
Respectively take 3 immunoaffinity......
Related standard:   GB 5009.22-2016  GB 5009.25-2016
   
 
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