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GB 5009.22-2016 English PDF (GB5009.22-2016)

GB 5009.22-2016 English PDF (GB5009.22-2016)

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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

This standard specifies the method for determination of 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 the isotope dilution liquid chromatographytandem mass spectrometry, which is suitable for the determination of AFT B1, AFT B2, AFT G1 and AFT G2 in cereals and their products, beans and their products, nuts and seeds, fats and their products, seasonings, infant formula and infant complementary foods.
GB 5009.22-2016
GB
NATIONAL STANDARD OF THE
PEOPLE 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 ??L 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 ??L 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 ??m 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 ??m), or
equivalent;
d) Flow rate. 1.0 mL/min;
e) Column temperature. 40 ??C;
f) Injection volume. 50 ??L.
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 ??m 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 ??m 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 ??m 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 ??m 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 ??m 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 ??m), or equivalent;
d) Flow rate. 0.3 mL/min;
e) Column temperature. 40 ??C;
f) Injection volume. 10 ??L;
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 ??g/kg, the detection limit of AFT B2 is 0.01 ??g/kg, the detection limit of AFT G1 is 0.03 ??g/kg, the detection limit of AFT G2 is 0.01 ??g/kg; for the non-derivatization, the detection limit of AFT B1 is 0.02 ??g/kg, the detection limit of AFT B2 is 0.003 ??g/kg, the detection limit of AFT G1 is 0.02 ??g/kg, the detection limit of AFT G2 is 0.003 ??g/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 ??g/kg, the limit of quantification of AFT B2 is 0.03 ??g/kg, the limit of quantification of AFT G1 is 0.1 ??g/kg, the limit of quantification of AFT G2 is 0.03 ??g/kg; for the non- derivatization. the limit of quantifica...

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