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GB 5009.22-2016: Determination of aflatoxin B1, B2, G1, G2, M1, M2 content in milk and milk powder -- HPLC-fluorescence detection method
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
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 conta...