GB 5009.240-2023 PDF in English
GB 5009.240-2023 (GB5009.240-2023) PDF English
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National food safety standard - Determination of fumonisin in foods
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GB 5009.240-2023: PDF in English GB 5009.240-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of
Fumonisin in Foods
ISSUED ON. SEPTEMBER 6, 2023
IMPLEMENTED ON. MARCH 6, 2024
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
Method I - Immunoaffinity Column Purification - Post-column Derivatization High-
performance Liquid Chromatography... 4
2 Principle... 4
3 Reagents and Materials... 4
4 Instruments and Equipment... 7
5 Analytical Procedures... 7
6 Expression of Analysis Results... 9
7 Precision... 10
8 Others... 10
Method II - High-performance Liquid Chromatography - Tandem Mass Spectrometry
... 10
9 Principle... 10
10 Reagents and Materials... 11
11 Instruments and Equipment... 13
12 Analytical Procedures... 14
13 Expression of Analysis Results... 18
14 Precision... 19
15 Others... 19
Method III - Immunoaffinity Column Purification - Pre-column Derivatization High-
performance Liquid Chromatography... 19
16 Principle... 19
17 Reagents and Materials... 19
18 Instruments and Equipment... 22
19 Analytical Procedures... 22
20 Expression of Analysis Results... 25
21 Precision... 25
22 Others... 25
Appendix A Post-column Derivatization - High-performance Liquid Chromatogram 27
Appendix B MRM Mass Chromatogram... 28
Appendix C Pre-column Derivatization - High-performance Liquid Chromatogram. 31
Appendix D Column Capacity and Column Recovery Verification Methods... 32
National Food Safety Standard - Determination of
Fumonisin in Foods
1 Scope
This Standard specifies the immunoaffinity column purification - post-column derivatization
high-performance liquid chromatography, high-performance liquid chromatography - tandem
mass spectrometry and immunoaffinity column purification - pre-column derivatization high-
performance liquid chromatography for the determination of fumonisin in foods.
This Standard is applicable to the determination of fumonisin B1, fumonisin B2 and fumonisin
B3 (hereinafter referred to as FB1, FB2 and FB3) in cereals and products, cereal supplementary
foods for infants and young children, and vegetable oils.
Method I - Immunoaffinity Column Purification - Post-
column Derivatization High-performance Liquid
Chromatography
2 Principle
The specimen is extracted, purified by an immunoaffinity column, separated by C18 reversed-
phase chromatography column, derivatized by o-phthalaldehyde, detected by fluorescence
detector, and quantified by the external standard method.
3 Reagents and Materials
Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and
the water is Grade-1 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH). chromatographically pure.
3.1.2 Acetonitrile (CH3CN). chromatographically pure.
3.1.3 Formic acid (HCOOH). chromatographically pure.
3.1.4 Acetic acid (CH3COOH).
3.1.5 Sodium hydroxide (NaOH).
3.1.6 Sodium chloride (NaCl).
3.1.7 Disodium hydrogen phosphate (Na2HPO4).
3.1.8 Potassium dihydrogen phosphate (KH2PO4).
3.1.9 Potassium chloride (KCl).
3.1.10 Borax (Na2B4O7 10H2O).
3.1.11 2-mercaptoethanol (C2H6OS).
3.1.12 o-phthalaldehyde (C8H6O, OPA).
3.1.13 Tween-20 (C58H114O26).
3.1.14 Hydrochloric acid (HCl).
3.2 Preparation of Reagents
3.2.1 Formic acid - water solution (0.1%). accurately transfer-take 1 mL of formic acid, use
water to dilute to 1,000 mL and evenly mix it.
3.2.2 Acetonitrile - water solution (50 + 50). respectively measure-take 500 mL of acetonitrile
and 500 mL of water, and evenly mix them.
3.2.3 Acetonitrile - water solution (20 + 80). respectively measure-take 20 mL of acetonitrile
and 80 mL of water, and evenly mix them.
3.2.4 Methanol - acetic acid solution (98 + 2). accurately transfer-take 2 mL of acetic acid, use
methanol to dilute to 100 mL and evenly mix it.
3.2.5 Sodium hydroxide solution (2 mol/L). accurately weigh-take 8.0 g of sodium hydroxide,
add 50 mL of water to dissolve it; after cooling, use water to dilute it to 100 mL and evenly mix
it.
3.2.6 Phosphate buffer solution (PBS). weigh-take 8.0 g of sodium chloride, 1.2 g of disodium
hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate and 0.2 g of potassium chloride;
use 980 mL of water to dissolve it, use hydrochloric acid to adjust pH to 7.4, use water to dilute
to 1,000 mL and evenly mix it.
3.2.7 Tween-20/PBS solution (0.1%). weigh-take 1.0 g of Tween-20, add phosphate buffer
solution, dilute to 1,000 mL and evenly mix it.
3.2.8 Borax solution (0.05 mol/L, pH 10.5). weigh-take 19.1 g of borax, dissolve it in 980 mL
of water, use sodium hydroxide solution to adjust pH to 10.5, use water to dilute to 1,000 mL
and evenly mix it.
3.2.9 Derivatization solution. weigh-take 0.5 g of o-phthalaldehyde, dissolve it in 20 mL of
methanol, use borax solution (0.05 mol/L, pH 10.5) to dilute to 500 mL, add 500 L of 2-
mercaptoethanol and evenly mix it. After filtration, put it into a brown bottle; store it at room
temperature away from light. It shall remain valid for 1 week.
3.3 Reference Materials
3.3.1 Fumonisin B1 (C34H59NO15, FB1, CAS No.. 116355-83-0), purity 95%, or a standard
substance certified by the state and awarded a standard substance certificate.
3.3.2 Fumonisin B2 (C34H59NO14, FB2, CAS No.. 116355-84-1), purity 95%, or a standard
substance certified by the state and awarded a standard substance certificate.
3.3.3 Fumonisin B3 (C34H59NO14, FB3, CAS No.. 136379-59-4), purity 95%, or a standard
substance certified by the state and awarded a standard substance certificate.
3.4 Preparation of Standard Solutions
3.4.1 Standard stock solutions (100 g/mL). respectively and accurately weigh-take 10 mg
(accurate to 0.01 mg) of FB1, FB2 and FB3 into small beakers, use acetonitrile - water solution
(50 + 50) to dissolve them, and respectively transfer to 100 mL volumetric flasks. Use
acetonitrile - water solution (50 + 50) to reach a constant volume to the scale. Store them at 18
C away from light. They shall remain valid for 6 months.
3.4.2 Mixed standard stock solution. accurately transfer-take 1.0 mL of FB1 standard stock
solution, 0.5 mL of FB2 standard stock solution and 0.5 mL of FB3 standard stock solution into
the same 10 mL volumetric flask; add acetonitrile - water solution (50 + 50) to dilute to the
scale. The mass concentration of FB1 is 10 g/mL, and the mass concentration of FB2 and FB3
is 5 g/mL. Store it at 18 C away from light. It shall remain valid for 6 months.
3.4.3 Mixed standard working solution. accurately transfer-take 1.0 mL of the mixed standard
stock solution into a 10.0 mL volumetric flask, add acetonitrile - water solution (50 + 50) to
dilute it and reach a constant volume to the scale. The mass concentration of FB1 is 1 g/mL,
and the mass concentration of FB2 and FB3 is 0.5 g/mL. Store it at 4 C away from light. It
shall remain valid for 6 months.
3.4.4 Mixed standard series of working solutions. accurately transfer-take the mixed standard
working solution, use acetonitrile - water solution (20 + 80) to dilute it, and prepare mixed
standard series of working solutions respectively with a FB1 mass concentration of 20.0 ng/mL,
80.0 ng/mL, 160 ng/mL, 240 ng/mL, 320 ng/mL, 400 ng/mL and 480 ng/mL, and a FB2 and
FB3 mass concentration of 10.0 ng/mL, 40.0 ng/mL, 80.0 ng/mL, 120 ng/mL, 160 ng/mL, 200
ng/mL and 240 ng/mL. Prepare them right before use.
3.5 Materials
3.5.1 Immunoaffinity column (see Appendix D for column capacity and column recovery
verification methods).
NOTE. before use, the column capacity and column recovery rate of each batch of affinity columns
5.2.2 Vegetable oils
The extraction of vegetable oils shall be performed in accordance with the steps in 5.2.1.The
extracting solution is the lower layer.
5.3 Specimen Purification
5.3.1 Cereal supplementary foods for infants and young children
Accurately transfer-take 5.0 mL of the extracting solution, add 45.0 mL of Tween-20/PBS
solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take the
supernatant and pass it all through the immunoaffinity column, and control the flow rate to 1
mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to
successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid
solution to elute the immunoaffinity column in three times; collect and combine the eluent. At
55 C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve
the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect
it in a sampling bottle and reserve it for testing.
5.3.2 Cereals and products, and vegetable oils
Accurately transfer-take 0.5 mL of the extracting solution, add 4.5 mL of Tween-20/PBS
solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take the
supernatant and pass it all through the immunoaffinity column, and control the flow rate to 1
mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to
successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid
solution to elute the immunoaffinity column in three times; collect and combine the eluent. At
55 C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve
the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect
it in a sampling bottle and reserve it for testing.
NOTE. since the immunoaffinity column operating procedures provided by different manufacturers
may be different, during actual operation, please refer to the operating instructions and
procedures provided by the manufacturer.
5.4 Reference Conditions of Instrument
5.4.1 Chromatographic column. C18, particle size 5 m, 4.6 250 mm, or equivalent.
5.4.2 Detection wavelength. excitation wavelength 335 nm; emission wavelength 440 nm.
5.4.3 Mobile phase. A. formic acid - water solution (0.1%); B. methanol. Gradient elution. The
elution procedure is shown in Table 1.
5.4.4 Mobile phase flow rate. 0.8 mL/min.
5.4.5 Derivative solution flow rate. 0.4 mL/min.
10.5 Preparation of Isotope Internal Standard Solution
10.5.1 Mixed isotope standard stock solution. accurately transfer-take 1 mL of 13C34-FB1 (25
g/mL), 13C34-FB2 (10 g/mL) and 13C34-FB3 (10 g/mL) into the same 10 mL volumetric flask.
Add acetonitrile - water solution (50 + 50) to dilute it and reach a constant volume to the scale.
The mass concentration of 13C34-FB1 is 2.5 g/mL, and the mass concentration of 13C34-FB2 and
13C34-FB3 is respectively 1 g/mL. Store it at 18 C away from light. It shall remain valid for
6 months.
10.5.2 Mixed isotope standard working solution. accurately transfer-take 1.0 mL of the mixed
isotope standard stock solution into a 10 mL volumetric flask, add acetonitrile - water solution
(50 + 50) to dilute it and reach a constant volume to the scale. The mass concentration of 13C34-
FB1 is 250 ng/mL, and the mass concentration of 13C34-FB2 and 13C34-FB3 is respectively 100
ng/mL. Store it at 4 C away from light. It shall remain valid for 6 months.
10.6 Preparation of Mixed Standard Series of Working Solutions
Accurately transfer-take the mixed standard working solution, use acetonitrile - water solution
(20 + 80) to dilute it, and add the mixed isotope standard working solution. Thus, mixed
standard series of working solutions with a FB1 mass concentration of 20.0 ng/mL, 80.0 ng/mL,
160 ng/mL, 240 ng/mL, 320 ng/mL, 400 ng/mL and 480 ng/mL, and a FB2 and FB3 mass
concentration of 10.0 ng/mL, 40.0 ng/mL, 80.0 ng/mL, 120 ng/mL, 160 ng/mL, 200 ng/mL and
240 ng/mL are prepared. In each standard working solution, the mass concentration of 13C34-
FB1, 13C34-FB2 and 13C34-FB3 is respectively 50.0 ng/mL, 20.0 ng/mL and 20.0 ng/mL. Prepare
them right before use.
10.7 Materials
10.7.1 Immunoaffinity column (see Appendix D for column capacity and column recovery
verification methods).
NOTE. before use, the column capacity and column recovery rate of each batch of affinity columns
need to be verified.
10.7.2 Strong anion exchange solid-phase extraction column (6 mL, 500 mg).
10.7.3 Microporous filter membrane. 0.22 m, organic type.
11 Instruments and Equipment
11.1 High-performance liquid chromatograph - tandem mass spectrometer. equipped with
electrospray ion source.
11.2 Balance. with a division value of 0.01 g and 0.01 mg.
11.3 Homogenizer. with a rotation speed 2,000 r/min.
11.4 Oscillator (with a rotation speed 1,000 r/min) or ultrasonic extraction instrument (with a
power 500 W).
11.5 Centrifuge. with a rotation speed 4,000 r/min.
11.6 Nitrogen blower.
12 Analytical Procedures
12.1 Specimen Preparation
For cereals and products, cereal supplementary foods for infants and young children, the
sampling size shall be not lower than 1 kg; when the mass of sample is less than 1 kg, all
specimens shall be taken. Use a high-speed pulverizer to pulverize it, and the fineness of
pulverization shall be less than 1 mm. After evenly mix it, store in a clean container, seal and
store at 4 C away from light.
For vegetable oils, the sampling size shall be not lower than 1 kg; when the mass of sample is
less than 1 kg, all specimens shall be taken. After evenly mix it, store in a clean container, seal
and store at 4 C away from light.
During the specimen preparation, sample contamination or changes in fumonisin content shall
be prevented.
12.2 Specimen Extraction
12.2.1 Cereals and products, cereal supplementary foods for infants and young children
Accurately weigh-take 20 g (accurate to 0.01 g) of specimen into a 250 mL conical flask,
accurately add 100 mL of acetonitrile - water (50 + 50) extracting solution, conduct ultrasonic
or oscillating extraction for 20 minutes, transfer 20 mL of the extracting solution into a 50 mL
centrifuge tube. At 4,000 r/min, centrifuge it for 5 minutes and reserve it for purification.
12.2.2 Vegetable oils
The extraction of vegetable oils shall be the same as 12.2.1.The extracting solution is the lower
layer.
12.3 Specimen Purification
12.3.1 Immunoaffinity column purification
12.3.1.1 Cereal supplementary foods for infants and young children
Accurately transfer-take 5.0 mL of the extracting solution, add 200 L of the mixed isotope
standard working solution and 45.0 mL of Tween-20/PBS solution for dilution, evenly mix it.
At 4,000 r/min, centrifuge it for 10 minutes, take all the supernatant and pass it through the
immunoaffinity column, and control the flow rate to 1 mL/min ~ 2 mL/min. Then, use 10 mL
of PBS buffer solution and 10 mL of water to successively rinse the immunoaffinity column.
Then, use 3 mL of methanol - acetic acid solution (98 + 2) to elute the immunoaffinity column
in three times; combine the eluent. At 55 C, use nitrogen to blow-dry it; add 1 mL of
acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing
it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing.
12.3.1.2 Cereals and products, and vegetable oils
Accurately transfer-take 0.5 mL of the extracting solution, add 200 L of the mixed isotope
standard working solution and 4.5 mL of Tween-20/PBS solution for dilution and evenly mix
it. At 4,000 r/min, centrifuge it for 10 minutes, take all the supernatant and pass it through the
immunoaffinity column, and control the flow rate to 1 mL/min ~ 2 mL/min. Then, use 10 mL
of PBS buffer solution and 10 mL of water to successively rinse the immunoaffinity column.
Then, use 3 mL of methanol - acetic acid solution to elute the immunoaffinity column in three
times; collect and combine the eluent. At 55 C, use nitrogen to blow-dry it; add 1 mL of
acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing
it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing.
12.3.2 Strong anion exchange solid-phase extraction purification column
12.3.2.1 Cereal supplementary foods for infants and young children
Accurately transfer-take 5.0 mL of the extracting solution, add 200 L of the mixed isotope
standard working solution and 15.0 mL of methanol - water solution (60 + 20) for dilution, and
evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes. Take all the supernatant and pass it
through a strong anion exchange solid-phase extraction column (before use, successively use
6.0 mL of methanol and 6.0 mL of water to activate it), and control the flow rate to 1 mL/min
~ 2 mL/min. Use 8 mL of methanol - water solution (60 + 20) and 3 mL of methanol to
successively rinse it, and use 10 mL of methanol - acetic acid solution (99 + 1) to elute it; collect
the eluent. At 55 C, use nitrogen to blow-dry it. Add 1 mL of acetonitrile - water solution (20
+ 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter
membrane, collect it in a sampling bottle and reserve it for testing.
12.3.2.2 Cereals and products, and vegetable oils
Accurately transfer-take 0.5 mL of the extracting solution, add 200 L of the mixed isotope
standard working solution and 5.0 mL of methanol - water solution (60 + 20) for dilution, and
evenly mix it. At 4,000 r/min, centrifuge it for 5 minutes. Take all the supernatant and pass it
through a strong anion exchange solid-phase extraction column (before use, successively use
6.0 mL of methanol and 6.0 mL of water to activate it), and control the flow rate to 1 mL/min
~ 2 mL/min. Use 8 mL of methanol - water solution (60 + 20) and 3 mL of methanol to
successively rinse it, and use 10 mL of methanol - acetic acid solution (99 + 1) to elute it; collect
the eluent. At 55 C, use nitrogen to blow-dry it. Add 1 mL of acetonitrile - water solution (20
+ 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter
membrane, collect it in a sampling bottle and reserve it for testing.
hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate and 0.2 g of potassium chloride;
use 980 mL of water to dissolve it, then, use hydrochloric acid to adjust pH to 7.4.Finally, use
water to dilute to 1,000 mL and evenly mix it.
17.2.7 Tween-20/PBS solution (0.1%). weigh-take 1.0 g of Tween-20, add phosphate buffer
solution, dilute to 1,000 mL and evenly mix it.
17.2.8 Borax solution (0.1 mol/L). weigh-take 3.8 g of borax, use water to dissolve it and dilute
to 100 mL, and evenly mix it.
17.2.9 Derivatization solution. weigh-take 25 mg of o-phthalaldehyde, dissolve it in 1 mL of
methanol, use borax solution (0.1 mol/L) to dilute to 50 mL, add 50 L of 2-mercaptoethanol
and evenly mix it. After filtration, put it into a brown bottle; store it at room temperature away
from light. It shall remain valid for 1 week.
17.3 Reference Materials
17.3.1 Fumonisin B1 (C34H59NO15, FB1, CAS No.. 116355-83-0), purity 95%, or a standard
substance certified by the state and awarded a standard substance certificate.
17.3.2 Fumonisin B2 (C34H59NO14, FB2, CAS No.. 116355-84-1), purity 95%, or a standard
substance certified by the state and awarded a standard substance certificate.
17.3.3 Fumonisin B3 (C34H59NO14, FB3, CAS No.. 136379-59-4), purity 95%, or a standard
substance certified by the state and awarded a standard substance certificate.
17.4 Preparation of Standard Solutions
17.4.1 Standard stock solutions (100 g/mL). respectively and accurately weigh-take 10 mg
(accurate to 0.01 mg) of FB1, FB2 and FB3 into small beakers, use acetonitrile - water solution
(50 + 50) to dissolve them, and respectively transfer to 100 mL volumetric flasks. Use
acetonitrile - water solution (50 + 50) to reach a constant volume to the scale. Store them at 18
C away from light. They shall remain valid for 6 months.
17.4.2 Mixed standard stock solution. accurately transfer-take 1.0 mL of FB1 standard stock
solution, 0.5 mL of FB2 standard stock solution and 0.5 mL of FB3 standard stock solution into
the same 10 mL volumetric flask; add acetonitrile - water solution (50 + 50) to dilute to the
scale. The mass concentration of FB1 is 10 g/mL, and the mass concentration of FB2 and FB3
is 5 g/mL. Store it at 18 C away from light. It shall remain valid for 6 months.
17.4.3 Mixed standard working solution. accurately transfer-take 1.0 mL of the mixed standard
stock solution into a 10.0 mL volumetric flask, add acetonitrile - water solution (50 + 50) to
dilute it and reach a constant volume to the scale. The mass concentration of FB1 is 1.0 g/mL,
and the mass concentration of FB2 and FB3 is 0.5 g/mL. Store it at 4 C away from light. It
shall remain valid for 6 months.
17.4.4 Mixed standard series of working solutions. accurately transfer-take the mixed standard
During the specimen preparation, sample contamination or changes in fumonisin content shall
be prevented.
19.2 Specimen Extraction
19.2.1 Cereals and products, cereal supplementary foods for infants and young children
Accurately weigh-take 20 g (accurate to 0.01 g) of specimen into a 250 mL conical flask,
accurately add 100 mL of acetonitrile - water (50 + 50) extracting solution, conduct ultrasonic
or oscillating extraction for 20 minutes, transfer 20 mL of the extracting solution into a 50 mL
centrifuge tube. At 4,000 r/min, centrifuge it for 10 minutes and reserve it for purification.
19.2.2 Vegetable oils
The extraction of vegetable oils shall be performed in accordance with the steps in 19.2.1.The
extracting solution is the lower layer.
19.3 Specimen Purification
19.3.1 Cereal supplementary foods for infants and young children
Accurately transfer-take 5.0 mL of the extracting solution, add 45.0 mL of Tween-20/PBS
solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take all the
supernatant and pass it through the immunoaffinity column, and control the flow rate to 1
mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to
successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid
solution (98 + 2) to elute the immunoaffinity column in three times; collect the eluent. At 55
C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve
the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect
it in a sampling bottle and reserve it for testing.
19.3.2 Cereals and products, and vegetable oils
Accurately transfer-take 0.5 mL of the extracting solution, add 4.5 mL of Tween-20/PBS
solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take all the
supernatant and pass it through the immunoaffinity column, and control the flow rate to 1
mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to
successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid
solution (98 + 2) to elute the immunoaffinity column in three times; collect the eluent. At 55
C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve
the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect
it in a sampling bottle and reserve it for testing.
NOTE. since the immunoaffinity column operating procedures provided by different manufacturers
may be different, during actual operation, please refer to the operating instructions and
procedures provided by the manufacturer.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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