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GB 5009.22-2016

Chinese Standard: 'GB 5009.22-2016'
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GB 5009.22-2016English300 Add to Cart 0--15 minutes. Auto immediate delivery. Determination of aflatoxin B1, B2, G1, G2, M1, M2 content in milk and milk powde--HPLC-fluorescence detection method Valid GB 5009.22-2016
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Detail Information of GB 5009.22-2016; GB5009.22-2016
Description (Translated English): Determination of aflatoxin B1, B2, G1, G2, M1, M2 content in milk and milk powde. HPLC-fluorescence detection method
Sector / Industry: National Standard
Classification of Chinese Standard: X04
Classification of International Standard: 67.050
Word Count Estimation: 37,346
Date of Issue: 2016-12-23
Date of Implementation: 2017-06-23
Older Standard (superseded by this standard): GB 5009.24-2010 Partial; GB/T 18979-2003; GB/T 23212-2008 Partial; GB/T 5009.22-2003; GB/T 5009.23-2006; NY/T 1286-2007; SN 0339-1995; SN/T 1101-2002; SN/T 1664-2005 Partial; SN/T 1736-2006
Regulation (derived from): National Health and Family Planning Commission Notice No.17 of 2016
Issuing agency(ies): National Health and Family Planning Commission of the People Republic of China, State Administration of Food and Drug Administration
Summary: This standard specifies the determination method for aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2 (hereinafter referred to as AFT B1, AFT B2, AFT G1 and AFT G2) in food. The first method of this standard is isotope dilution liquid chromatography-tandem mass spectrometry for cereals and their products, beans and their products, nuts and seeds, oils and fats and their products, condiments, infant formula and infants and young children Determination of AFT B1, AFT B2, AFT G1 and AFT G2 in Food. The second method of this standard is a high performance liquid chromatography pre-column derivatization method for cereals and their products, legumes and their products, nuts and seeds, oils and fats and their products, condiments, infant formula and infants and young children Food

GB 5009.22-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of B-
group and G-group Aflatoxins in Foods
食品安全国家标准
食品中黄曲霉素 B族和 G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 
2 Principle ... 6 
3 Reagents and materials ... 6 
4 Instruments and equipment... 8 
5 Analytical procedures ... 9 
6 Expression of analytical results ... 15 
7 Precision ... 15 
8 Others ... 16 
9 Principle ... 16 
10 Reagents and materials ... 16 
11 Instruments and equipment ... 18 
12 Analytical procedures ... 19 
13 Expression of analytical results ... 21 
14 Precision ... 22 
15 Others ... 22 
16 Principle ... 23 
17 Reagents and materials ... 23 
18 Instruments and equipment... 25 
19 Analytical procedures ... 27 
20 Expression of analytical results ... 31 
21 Precision ... 32 
22 Others ... 32 
23 Principle ... 33 
24 Reagents and materials ... 33 
25 Instruments and equipment... 33 
26 Analytical procedures ... 34 
27 Expression of analytical results ... 34 
28 Precision ... 35 
29 Others ... 35 
30 Principle ... 36 
31 Reagents and materials ... 36 
32 Instruments and equipment... 38 
33 Analytical steps ... 38 
34 Precision ... 46 
35 Others ... 46 
Appendix A Calibration method of standard concentration of AFT B1, AFT B2,
AFT G1 and AFT G2 ... 47 
Appendix B Method of verification of immunoaffinity column ... 49 
Appendix C Tandem mass spectrometry... 50 
Appendix D Liquid chromatogram ... 55 
Appendix E Determination method of quality of enzyme-linked immunosorbent
kit ... 59 
Method I. Isotope dilution liquid chromatography-
tandem mass spectrometry
2 Principle
The aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2 in the specimen are
extracted by acetonitrile-water solution or methanol-water solution. After the
extract is diluted by the phosphate buffer solution which contains 1% Triton X-
100 (or Tween-20) (if necessary, it is initially purified by the aflatoxin solid-phase
purification column), through purification and enrichment by immunoaffinity
column, the purification liquid is concentrated, constant-volume, filtered,
separated by liquid chromatography, tested by tandem mass spectrometry, then
subjected to quantification by isotope internal standard method.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical
grade, the water is the grade I water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Acetonitrile (CH3CN). Chromatographically pure.
3.1.2 Methanol (CH3OH). Chromatographically pure.
3.1.3 Ammonium acetate (CH3COONH4). Chromatographically pure.
3.1.4 Sodium chloride (NaCl).
3.1.5 Disodium hydrogen phosphate (Na2HPO4).
3.1.6 Potassium dihydrogen phosphate (KH2PO4).
3.1.7 Potassium chloride (KCl).
3.1.8 Hydrochloric acid (HCl).
3.1.9 Triton X-100 [C14H22O(C2H4O)n] (or Tween-20, C58H114O26).
3.2 Preparation of reagents
3.2.1 Ammonium acetate solution (5 mmol/L). WEIGH 0.39 g of ammonium
acetate; DISSOLVE it in water; USE water to dilute it to 1000 mL; MIX it
uniformly.
of exposure to severe poisons.
5.1 Preparation of sample
5.1.1 Liquid samples (vegetable oil, soy sauce, vinegar, etc.)
The sampling amount shall be greater than 1 L. For packaging samples such
as bags and bottles, it shall collect at least 3 packages (same batch or number).
All liquid samples shall be mixed by a homogenizer in a container, any 100 g
(mL) of sample is taken for testing.
5.1.2 Solid samples (cereals and their products, nuts and seeds, cereal
supplements for infants, etc.)
The sampling amount shall be greater than 1 kg. It shall be crushed by high-
speed pulverizer and sieved to make the particle size less than the 2 mm
aperture test sieve. It is evenly mixed and then reduced to 100 g, stored in the
sample bottle, sealed for preservation, to prepare for testing.
5.1.3 Semi-fluid (fermented bean curd, fermented soybean, etc.)
The sampling amount shall be greater than 1 kg (L). For packaging samples
such as bags and bottles, it shall collect at least 3 packages (same batch or
number). Then it is crushed and mixed uniformly by the tissue crusher, stored
in sample bottle, sealed for preservation, to prepare for testing.
5.2 Extraction of sample
5.2.1 Liquid sample
5.2.1.1 Vegetable oils
WEIGH 5 g of specimen (accurate to 0.01 g) in a 50 mL centrifuge tube; ADD
100 μL of isotope internal standard working solution (3.4.3); OSCILLATE to mix
it uniformly; LET it be standing for 30 min. ADD 20 mL of acetonitrile-water
solution (84 + 16) or methanol-water solution (70 + 30); VORTEX to mix it
uniformly; PLACE it in the ultrasonic/vortex oscillator or shaker to oscillate it for
20 min (or homogenized by homogenizer for 3 min). CENTRIFUGE it at 6000
r/min for 10 min, TAKE the supernatant to prepare for use.
5.2.1.2 Soy sauce, vinegar
WEIGH 5 g of specimen (accurate to 0.01 g) into a 50 mL centrifuge tube; ADD
125 μL of isotope internal standard working solution; OSCILLATE to mix it; LET
it be standing for 30 min. USE acetonitrile or methanol to make its volume reach
to 25 mL (accurate to 0.1 mL); VORTEX to mix it uniformly; PLACE it in the
ultrasonic/vortex oscillator or shaker to oscillate it for 20 min (or homogenized
by homogenizer for 3 min). CENTRIFUGE it at 6000 r/min for 10 min (or
RESTORE the immunoaffinity column which is preserved at low temperature to
room temperature.
5.3.1.3 Purification of specimen
After the original liquid in the immunoaffinity column is exhausted, PIPETTE the
above sample solution into a 50 mL injection syringe tube; ADJUST the
dropping speed, to make the sample solution drop stably at the speed of 1
mL/min ~ 3 mL/min. After the sample solution drops completely, ADD 2 x 10 mL
of water into the injection syringe tube; RINSE the immunoaffinity column at a
steady flow rate. After the water dropping is finished, USE a vacuum pump to
empty the immunoaffinity column. REMOVE it from the vacuum system; PLACE
a 10 mL graduated test tube in the lower part of the immunoaffinity column;
TAKE off the 50 mL injection syringe tube; ADD 2 × 1 mL methanol to elute the
immunoaffinity column; CONTROL the dropping speed at 1 mL/min ~ 3 mL/min;
then USE the vacuum pump to empty the immunoaffinity column; COLLECT all
the eluate into the test tube. At 50 °C, USE nitrogen to slowly blow the eluate
to almost dry; ADD 1.0 mL of initial mobile phase; VORTEX it for 30 s to dissolve
the residue; USE the 0.22 μm membrane to filter it; COLLECT the filtrate in the
sample bottle to prepare for sample injection.
5.3.2 Simultaneous use of aflatoxin solid-phase purification column and
immunoaffinity column (for complex substrates such as Chinese red
pepper, black pepper and chili pepper)
5.3.2.1 Purification of purification column
PIPETTE an appropriate amount of supernatant; FOLLOW the operation
instruction of purification column to purify it; COLLECT all the purified solution.
5.3.2.2 Purification of immunoaffinity column
USE a graduated pipette to accurately pipette 4 mL of the above purified
solution; ADD 46 mL of PBS which contains 1% Trition X-100 (or Tween-20)
[when extracted by methanol-water solution, ADD 23 mL of PBS which contains
1% Trition X-100 (or Tween-20)], MIX it uniformly. PROCESS it in accordance
with 5.3.1.2 and 5.3.1.3.
Note. Fully-automatic (online) or semi-automatic (offline) solid-phase extraction
instruments may be used after optimizing the operating parameters.
5.4 Reference conditions for liquid chromatography
The reference conditions for liquid chromatography are listed below.
a) Mobile phase. phase A. 5 mmol/L ammonium acetate solution; phase B.
acetonitrile-methanol solution (50 + 50);
12 Analytical procedures
12.1 Preparation of sample
12.1.1 Liquid samples (vegetable oil, soy sauce, vinegar, etc.)
The sampling amount shall be greater than 1 L. For packaging samples such
as bags and bottles, it shall collect at least 3 packages (same batch or number).
All liquid samples shall be mixed by a homogenizer in a container, any 100 g
(mL) of sample is taken for testing.
12.1.2 Solid samples (cereals and their products, nuts and seeds, cereal
supplements for infants, etc.)
The sampling amount shall be greater than 1 kg. It shall be crushed by high-
speed pulverizer and sieved to make the particle size less than the 2 mm
aperture test sieve. It is evenly mixed and then reduced to 100 g, stored in the
sample bottle, sealed for preservation, to prepare for testing.
12.1.3 Semi-fluid (fermented bean curd, fermented soybean, etc.)
The sampling amount shall be greater than 1 kg (L). For packaging samples
such as bags and bottles, it shall collect at least 3 packages (same batch or
number). Then it is crushed and mixed uniformly by the tissue crusher, stored
in sample bottle, sealed for preservation, to prepare for testing.
12.2 Extraction of sample
12.2.1 Liquid sample
12.2.1.1 Vegetable oils
WEIGH 5 g of specimen (accurate to 0.01 g) in a 50 mL centrifuge tube; ADD
20 mL of acetonitrile-water solution (84 + 16) or methanol-water solution (70 +
30); VORTEX to mix it uniformly; PLACE it in the ultrasonic/vortex oscillator or
shaker to oscillate it for 20 min (or homogenized by homogenizer for 3 min).
CENTRIFUGE it at 6000 r/min for 10 min, TAKE the supernatant to prepare for
use.
12.2.1.2 Soy sauce, vinegar
WEIGH 5 g of specimen (accurate to 0.01 g) into a 50 mL centrifuge tube; USE
acetonitrile or methanol to make its volume reach to 25 mL (accurate to 0.1 mL);
VORTEX to mix it uniformly; PLACE it in the ultrasonic/vortex oscillator or
shaker to oscillate it for 20 min (or homogenized by homogenizer for 3 min).
CENTRIFUGE it at 6000 r/min for 10 min (or otherwise homogenized and then
filtered by glass-fiber filter paper), TAKE the supernatant to prepare for use.
The reference conditions for chromatography are listed below.
a) Mobile phase. phase A. water; phase B. acetonitrile-methanol solution (50
+ 50);
b) Gradient elution. 24% B (0 min ~ 6 min), 35% B (8.0 min ~ 10.0 min), 100%
B (10.2 min ~ 11.2 min), 24% B (11.5 min ~ 13.0 min);
c) Chromatography column. C18 column (column’s length 150 mm or 250 mm,
column’s inner diameter 4.6 mm; packing’s particle size 5.0 μm), or
equivalent;
d) Flow rate. 1.0 mL/min;
e) Column temperature. 40 °C;
f) Injection volume. 50 μL.
g) Detection wavelength. Excitation wavelength 360 nm; Emission
wavelength 440 nm;
h) Liquid chromatogram. See Figure D.1.
12.6 Determination of sample
12.6.1 Production of standard curve
The series standard working solution is subjected to sample injection testing in
the order from low to high concentration. USE the peak area as the ordinate;
USE the concentration as the abscissa to make plotting, to obtain the standard
curve regression equation.
12.6.2 Determination of sample solution
The response value of the compound to be tested in the sample solution to be
tested shall be within the linear range of the standard curve. The sample whose
concentration exceeds the linear range shall be subjected to sample injection
analysis again.
12.6.3 Blank test
DO not weigh the specimen; PERFORM the blank test in accordance with the
steps of 12.2, 12.3 and 12.4. It shall be confirmed that it does not contain
substances that interfere with the component to be tested.
13 Expression of analytical results
The residual amount of AFT B1, AFT B2, AFT G1 and AFT G2 in the specimen is
17.1.11 Electrochemical derivatization reagents. potassium bromide (KBr),
concentrated nitric acid (HNO3).
17.2 Preparation of reagent
17.2.1 Acetonitrile-water solution (84 + 16). TAKE 840 mL of acetonitrile; ADD
160 mL of water.
17.2.2 Methanol-water solution (70 + 30). TAKE 700 mL of methanol; ADD 300
mL of water.
17.2.3 Acetonitrile-water solution (50 + 50). TAKE 500 mL of acetonitrile; ADD
500 mL of water.
17.2.4 Acetonitrile-water solution (10 + 90). TAKE 100 mL of acetonitrile; ADD
900 mL of water.
17.2.5 Acetonitrile-methanol solution (50 + 50). TAKE 500 mL of acetonitrile;
ADD 500 mL of methanol.
17.2.6 Phosphate buffer solution (hereinafter referred to as PBS). WEIGH 8.00
g of sodium chloride, 1.20 g of disodium hydrogen phosphate (or 2.92 g of
disodium hydrogen phosphate dodecahydrate), 0.20 g of potassium dihydrogen
phosphate, 0.20 g of potassium chloride; USE 900 mL of water to dissolve it;
USE hydrochloric acid to adjust the pH to 7.4; USE water to make its volume
reach to 1000 mL.
17.2.7 The PBS which contains 1% Triton X-100 (or Tween-20). TAKE 10 mL of
Triton X-100; USE PBS to dilute it to 1000 mL.
17.2.8 The 0.05% iodine solution. WEIGH 0.1g of iodine; USE 20 mL of
methanol to dissolve it; ADD water to make its volume reach to 200 mL; USE
the 0.45 μm filter membrane to filter it; PREPARE it before use (only used for
iodine post-column derivatization).
17.2.9 The 5 mg/L pyridine tribromide aqueous solution. WEIGH 5 mg of
pyridine tribromide; DISSOLVE it into 1 L of water; USE the 0.45 μm filter
membrane to filter it; PREPARE it before use (only used for bromine post-
column derivatization).
17.3 Standard substance
17.3.1 AFT B1 standard substance (C17H12O6, CAS. 1162-65-8). Purity ≥ 98%,
or other standard substance which is nationally certified and awarded with the
standard substance certificate.
17.3.2 AFT B2 standard substance (C17H14O6, CAS. 7220-81-7). Purity ≥ 98%,
or other standard substance which is nationally certified and awarded with the
18.5 Balance. Sensitivity 0.01 g and 0.00001 g.
18.6 Vortex mixer.
18.7 High-speed homogenizer. speed 6500 r/min ~ 24000 r/min.
18.8 Centrifuge. Speed ≥ 6000 r/min.
18.9 Glass-fiber filter paper. Fast, high-load, which retains the 1.6 μm particles
in the liquid.
18.10 Solid-phase extraction device (equipped with vacuum pump).
18.11 Nitrogen-blowing instrument.
18.12 Liquid chromatograph. Equipped with a fluorescence detector (with a flow
cell of general volume or large volume).
Note. When it is equipped with a flow cell of large volume, it does not require
the post-column derivative of any model or any methods.
18.13 Liquid chromatogram column.
18.14 Photochemical post-column derivative device (for photochemical post-
column derivatization).
18.15 Solvent post-column derivative device (for iodine or bromine reagent
derivatization).
18.16 Electrochemical post-column derivative device (for electrochemical post-
column derivatization).
18.17 Immunoaffinity column. Capacity of AFT B1 column ≥ 200 ng, recovery
rate of AFT B1 column ≥ 80%, cross-reaction rate of AFT G2 ≥ 80% (see
Appendix B for verification method).
Note. Quality verification is required for each batch of immunoaffinity columns
before use.
18.18 Aflatoxin solid-phase purification column or functionally equivalent solid-
phase extraction column (hereinafter referred to as purification column). It is
used for the determination of samples of complex matrix.
18.19 Disposable microporous filter. Equipped with 0.22 μm microporous
membrane (before use, the selected membrane shall be tested by standard
solution to confirm that there is no adsorption).
18.20 Sieve. 1 mm ~ 2 mm aperture test sieve.
TAKE off the 50 mL injection syringe tube; ADD 2 × 1 mL methanol to elute the
immunoaffinity column; CONTROL the dropping speed at 1 mL/min ~ 3 mL/min;
then USE the vacuum pump to empty the immunoaffinity column; COLLECT all
the eluate into the test tube. At 50 °C, USE nitrogen to slowly blow the eluate
to almost dry; USE the initial mobile phase to make its volume reach to 1.0 mL;
VORTEX it for 30 s to dissolve the residue; USE the 0.22 μm membrane to filter
it; COLLECT the filtrate in the sample bottle to prepare for sample injection.
19.3.2 Simultaneous use of aflatoxin solid-phase purification column and
immunoaffinity column (for complex substrates such as Chinese red
pepper, black pepper and chili pepper)
19.3.2.1 Purification of purification column
PIPETTE an appropriate amount of supernatant; FOLLOW the operation
instruction of purification column to purify it; COLLECT all the purified solution.
19.3.2.2 Purification of immunoaffinity column
USE a graduated pipette to accurately pipette 4 mL of the above purified
solution; ADD 46 mL of PBS which contains 1% Trition X-100 (or Tween-20)
(such amount may be halved when extracted by methanol-water solution); MIX
it uniformly. PROCESS it in accordance with 19.4.1.3.
Note. Fully automatic (online) or semi-automatic (offline) solid-phase extraction
instruments may be used after optimizing operating parameters.
19.4 Reference conditions for liquid chromatography
19.4.1 Non-derivatization method (direct testing with large flow cell)
The reference conditions for the liquid chromatography are listed below.
a) Mobile phase. phase A, water; phase B, acetonitrile-methanol (50 + 50);
b) Equal-gradient elution conditions. A, 65%; B, 35%;
c) Chromatography column. C18 column (column’s length 100 mm, column’s
inner diameter 2.1 mm, packing’s particle size 1.7 μm), or equivalent;
d) Flow rate. 0.3 mL/min;
e) Column temperature. 40 °C;
f) Injection volume. 10 μL;
g) Excitation wavelength. 365 nm; emission wavelength. 436 nm (AFT B1,
AFT B2), 463 nm (AFT G1, AFT G2);
injection solution in accordance with the internal standard method in the
standard curve, in nanograms per milliliter (ng/mL);
V1 - The volume of specimen extract (based on the volume of the added
extract for plant oil, solid, semi-solid; based on the total constant volume for
soy sauce, vinegar), in milliliter (mL);
V3 - The final constant volume of the sample after subjected to purification
and elution by immunoaffinity column, in milliliters (mL);
V2 - The volume of the sample taken for the immunoaffinity column, in
milliliters (mL);
1000 - Conversion factor;
m - The weighing amount of specimen, in grams (g).
The calculation result retains three significant digits.
21 Precision
The absolute difference between two independent determinations obtained
under repeatability conditions shall not exceed 20% of the arithmetic mean.
22 Others
When 5 g of sample is weighed, for the post-column photochemical
derivatization, post-column bromine derivatization, post-column iodine
derivatization, post-column electrochemical derivatization, the detection limit of
AFT B1 is 0.03 μg/kg, the detection limit of AFT B2 is 0.01 μg/kg, the detection
limit of AFT G1 is 0.03 μg/kg, the detection limit of AFT G2 is 0.01 μg/kg; for the
non-derivatization, the detection limit of AFT B1 is 0.02 μg/kg, the detection limit
of AFT B2 is 0.003 μg/kg, the detection limit of AFT G1 is 0.02 μg/kg, the
detection limit of AFT G2 is 0.003 μg/kg;
For the post-column photochemical derivatization, post-column bromine
derivatization, post-column iodine derivatization, post-column electrochemical
derivatization. the limit of quantification of AFT B1 is 0.1 μg/kg, the limit of
quantification of AFT B2 is 0.03 μg/kg, the limit of quantification of AFT G1 is 0.1
μg/kg, the limit of quantification of AFT G2 is 0.03 μg/kg; for the non-
derivatization. the limit of quantification of AFT B1 is 0.05 μg/kg, the limit of
quantification of AFT B2 is 0.01 μg/kg, the limit of quantification of AFT G is 0.05
μg/kg, the limit of quantification of AFT G2 is 0.01 μg/kg.
25.8 Instruments required for the kit.
26 Analytical procedures
26.1 Preparation of sample
26.1.1 Liquid samples (fat and seasoning)
TAKE 100 g of the sample to be tested; SHAKE it uniformly; WEIGH 5.0 g of
sample into a 50 mL centrifuge tube; ADD the extract required for the kit;
FOLLOW the methods as described in the operation instruction on the kit to
perform testing.
26.1.2 Solid samples (cereals, nuts and special dietary foods)
WEIGH at least 100 g of the sample; USE a grinder to pulverize it; after
pulverization, MAKE the sample pass through the 1 mm ~ 2 mm aperture test
sieve. TAKE 5.0 g of the sample in a 50 mL centrifuge tube; ADD the extract
required for the kit; FOLLOW the methods as described in the operation
instruction on the kit to perform testing.
26.2 Testing of sample
The specimen (liquid) to be tested is quantitatively tested in accordance with
the operation procedure as described in the enzyme-linked immunosorbent
assay kit.
27 Expression of analytical results
27.1 Drawing of standard working curve for quantitative testing of
enzyme-linked immunosorbent assay kit
In accordance with the calculation method or computer software provided in the
operation instruction on the kit, DRAW the standard working curve in
accordance with the relationship between the concentration of the standard
substance and the change of absorbance.
27.2 Calculation of liquid concentration to be tested
In accordance with the calculation method or computer software provided in the
operation instruction on the kit, the absorbance of the liquid to be tested is
substituted into the formula as obtained in 27.1, to calculate the concentration
of the liquid to be tested (ρ).
27.3 Calculation of results
Method V - Thin-layer chromatography
30 Principle
After the sample is extracted, concentrated, and thin-layer separated, the
aflatoxin B1 produces blue-violet fluorescence under ultraviolet light
(wavelength 365 nm), the content is determined based on the minimum
detected amount of fluorescence as displayed on the thin-layer.
31 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical
grade and the water is the grade I water as specified in GB/T 6682.
31.1 Reagents
31.1.1 Methanol (CH3OH).
31.1.2 n-Hexane (C6H14).
31.1.3 Petroleum ether (boiling range 30 °C ~ 60 °C or 60 °C ~ 90 °C).
31.1.4 Chloroform (CHCl3).
31.1.5 Benzene (C6H6).
31.1.6 Acetonitrile (CH3CN).
31.1.7 Anhydrous ether (C2H6O).
31.1.8 Acetone (C3H6O).
Note. The above reagents shall be subjected to one reagent blank test before
the test. If it does not interfere with the determination, it can be used. Otherwise,
it needs to be re-steamed one by one.
31.1.9 Silica gel G. for thin-layer chromatography.
31.1.10 Trifluoroacetic acid (CF3COOH).
31.1.11 Anhydrous sodium sulfate (Na2SO4).
31.1.12 Sodium chloride (NaCl).
31.2 Preparation of reagent
solution stock solution in a 10 mL volumetric flask; ADD the benzene-
acetonitrile mixed solution to the mark; MIX it uniformly. Each millimeter of this
solution is equivalent to 1.0 μg of AFT B1. PIPETTE 1.0 mL of this dilution into
a 5 mL volumetric flask; ADD the benzene-acetonitrile mixed solution to dilute
it to the mark. Each millimeter of this solution is equivalent to 0.2 μg of AFT B1.
Then TAKE another 1.0 mL of AFT B1 standard sputum (0.2 μg/mL) into a 5 mL
volumetric flask; ADD the benzene-acetonitrile mixed solution to dilute it to the
mark. Each millimeter of this solution is equivalent to 0.04 μg of AFT B1.
32 Instruments and equipment
32.1 Round-hole sieve. 2.0 mm aperture sieve.
32.2 Small-sized pulverizer.
32.3 Electric oscillator.
32.4 Full-glass concentrator.
32.5 Glass plate. 5 cm × 20 cm.
32.6 Thin-layer plate applicator.
Note. Commercially available thin-layer plate which is suitable for testing of
aflatoxin testing may be used.
32.7 Developing tank. length 25 cm, width 6 cm, height 4 cm.
32.8 Ultra-violet lamp. 100 W ~ 125 W, equipped with a 365 nm optical filter.
32.9 Microinjector or haemochrome pipette.
33 Analytical steps
Caution. The entire operation shall be carried out in darkroom conditions.
33.1 Extraction of sample
33.1.1 Corn, rice, wheat, flour, dried potato, beans, peanuts, peanut butter,
etc.
33.1.1.1 Method A. WEIGH 20.00 g of crushed and sieved specimen (flour,
peanut butter do not need to be crushed); PLACE it in a 250 mL stoppered
conical flask; ADD 30 mL of n-hexane or petroleum ether as well as 100 mL of
aqueous methanol solution; APPLY a layer of water onto the stopper; COVER
it tightly to avoid leakage. OSCILLATE it for 30 min; LET it be standing for a
while; USE the folded fast filter paper to filter it into a separatory funnel. After
33.1.3 Soy sauce, vinegar
WEIGH 10.00 g of the specimen in a small beaker. To prevent emulsification
during the extraction, ADD 0.4 g of sodium chloride; TRANSFER it into a
separatory funnel; USE 15 mL of chloroform to rinse the beaker for several
times; COLLECT the rinsing solution into the separatory funnel. PERFORM the
subsequent steps from “SHAKE it for 2 min; LET it be standing for
delamination ...” in accordance with 33.1.1.1. Finally ADD 2.5 mL of benzene-
acetonitrile mixed solution. Each millimeter of this solution is equivalent to 4 g
of specimen.
Or WEIGH 10.00 g of specimen; PLACE it in a separatory funnel; then ADD 12
mL of methanol (because the volume of soy sauce is used instead of water, the
volume ratio of methanol to water is still about 55.45); USE 20 mL of chloroform
to extract it. PERFORM the subsequent steps from “SHAKE it for 2 min; LET it
be standing for delamination ...” in accordance with 33.1.1.1. Finally ADD 2.5
mL of benzene-acetonitrile mixed solution. Each millimeter of this solution is
equivalent to 4 g of specimen.
33.1.4 Dry sauces (including fermented soybean, fermented bean curd
products)
WEIGH 20.00 g of the uniformly ground specimen; PLACE it in a 250 mL
stoppered conical flask; ADD 20 mL of n-hexane or petroleum ether as well as
50 mL of aqueous methanol solution. SHAKE it for 30 min; LET it be standing
for a while; USE the folded fast qualitative filter paper to filter it. After the filtrate
is standing for delamination, TAKE 24 mL of methanol water layer (equivalent
to 8 g of specimen, including 8 g of dry sauce itself which contains about 4 mL
of water); PLACE it in a separatory funnel; ADD 20 mL of chloroform.
PERFORM the subsequent steps from “SHAKE it for 2 min; LET it be standing
for delamination ...” in accordance with 33.1.1.1. Finally ADD 2 mL of
benzene-acetonitrile mixed solution. Each millimeter of this solution is
equivalent to 4 g of specimen.
33.2 Determination
33.2.1 One-way developing method
33.2.1.1 Preparation of thin-layer plates
WEIGH about 3 g of silica gel G; ADD water equivalent to 2 times ~ 3 times of
silica gel; GRIND it hard for 1 min ~ 2 min until it is into a paste; immediately
POUR it into the applicator; COAT it into three 5 cm × 20 cm thin-layer plates
which have a thickness about 0.25 mm. After drying it in the air for about 15
min; ACTIVATE it at 100 °C for 2 h; TAKE it out; PRESERVE it in a desiccator.
Generally, it can be preserved for 2 d ~ 3 d. If it is placed for a long time, it can
be used after reactivation.
derivative is about 0.1. DROP two points to the left of the thin-layer plate
sequentially.
First point-drop. 10 μL of 0.04 μg/mL AFT B1 standard working solution.
Second point-drop. 20 μL of sample solution. ADD a small drop of trifluoroacetic
acid to the above two points, respectively; after reaction for 5 min, USE a hair
dryer to blow hot air against it for 2 min; LET the temperature of the hot air which
is blown to the thin-layer plate is not more than 40 °C; then ADD the following
two points onto the thin-layer plate.
Third point-drop. 10 μL of 0.04 μg/mL AFT B1 standard working solution.
Fourth point-drop. 20 μL of sample solution.
MAKE it develop again (same as 16.2.1.3); under ultraviolet light, OBSERVE
whether the sample solution produces the same derivative as the AFT B1
standard point. The third and fourth points on which the trifluoroacetic acid is
not added can be used as the derivative blank control for the sample solution
and the standard.
33.2.1.5 Quantification of dilution
If the fluorescence intensity of the AFT B1 fluorescent spot in the sample
solution is consistent with the fluorescence intensity of the lowest detected
amount of the AFT B1 standard point (0.0004 μg), the AFT B1 content in the
specimen is 5 μg/kg. If the fluorescence intensity in the sample solution is more
intense than the minimum detected amount, then based on its intensity estimate,
reduce the microliter number or otherwise add different microliter number after
diluting the sample solution, until the fluorescence intensity of the sample
solution point is consistent with that of the minimum detected amount. The
dropping pattern is as follows.
First point-drop. 10 μL of AFT B1 standard working solution (0.04 μg/mL)
Second point-drop. ADD 10 μL of sample solution dropwise in accordance with
the situation.
Third point-drop. ADD 15 μL of sample solution dropwise in accordance with
the situation.
Fourth point-drop. ADD 20 μL of sample solution dropwise in accordance with
the situation.
33.2.1.6 Calculation of results
The content of AFT B1 in the specimen is calculated in accordance with formula
(5).
another 10 μL of the AFT B1 standard use solution (0.04 μg/mL) to the sample
solution point on the second plate; ADD another 10 μL of 0.2 μg/mL AFT B1
standard use solution to the sample solution point on the third plate.
33.2.2.1.2 Development
33.2.2.1.2.1 Transverse development. Along the long side of the developing
tank, PLACE a glass bracket; ADD 10 mL of anhydrous ethanol; PLACE the
long side of the thin-layer plate to which sample is applied and which is close
to the standard point int the developing tank to develop it; after developing to
the plate’s end, TAKE it out to evaporate it dry. Or otherwise REPEAT
development again for 1 ~ 2 times if required, based on actual conditions.
33.2.2.1.2.2 Longitudinal deployment. USE the acetone-chloroform (8 + 92) to
develop the thin-layer plate which is evaporated dry to 10 cm ~ 12 cm. The ratio
of acetone to chloroform is self-regulating in accordance with different
conditions.
33.2.2.1.3 Observation and assessment results
Under the ultraviolet light, OBSERVE the first plate and the second plate; if the
minimum detected amount occurs at the second point of the second plate in the
position corresponding to the AFT B1 standard point, whilst the fluorescent point
does not occur at the same positions on the first plate and the second plate, the
content of AFT B1 in the specimen is below 5 μg/kg.
If the fluorescent points occur at the same position of the first plate and the
second plate, COMPARE the first plate with the third plate, to check whether
the fluorescent point at the same position of the second point of the third plate
and the second point of the first point overlaps with the AFT B1 standard point;
if they overlap, PERFORM the confirmation test. In the specific determination,
the first, second and third plates can be subjected to confirmation test at the
same time or in sequence. If it is subjected to confirmation test in sequence,
when the first plate is negative, the third plate can be exempted of such test. If
the first plate is positive, the second plate can be exempted of such test, TEST
the third plate directly.
33.2.2.1.4 Confirmation test
TAKE another two thin-layer plates; ADD 10 μL of AFT B1 standard use solution
(0.04 μg/mL) and 1 small drop of trifluoroacetic acid to the fourth and fifth plates
at a distance 0.8 cm ~ 1 cm from the left edge. At the position 2.8 cm ~ 3 cm
from the left edge, ADD 20 μL of sample solution and 1 small drop of
trifluoroacetic acid dropwise to the fourth plate; ADD 20 μL of the sample
solution, 10 μL of the AFT B1 standard use solution (0.04 μg/mL) and 1 small
drop of trifluoroacetic acid dropwise onto the fifth plate. After reacting for 5
m......
Related standard:   GB 5009.13-2017  GB 5009.14-2017
   
 
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