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GB 5009.205-2024 PDF in English


GB 5009.205-2024 (GB5009.205-2024) PDF English
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GB 5009.205-2024: PDF in English

GB 5009.205-2024
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Determination of toxic
equivalent of dioxins and their analogues in food
ISSUED ON: FEBRUARY 08, 2024
IMPLEMENTED ON: AUGUST 08, 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
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and apparatuses ... 7
5 Analysis steps ... 8
6 Expression of analysis results ... 17
7 Precision ... 20
8 Detection-limit ... 20
9 Others ... 20
10 Principle ... 21
11 Reagents and materials ... 21
12 Instruments and apparatuses ... 21
13 Procedure ... 21
14 Expression of analysis results ... 25
15 Precision ... 27
16 Quantitation-limit of the method ... 27
17 Others ... 27
Appendix A Name, CAS number, IUPAC number, toxic equivalency factor (TEF)
specified by WHO of 17 kinds of 2,3,7,8-substituted PCDD/Fs and 12 kinds of DL-
PCBs ... 28
Appendix B Standard solutions ... 29
Appendix C Technical requirements for determination methods ... 34
Appendix D Separation and purification process of fully automatic sample purification
system ... 41
Appendix E Standard solution chromatograms ... 44
National food safety standard - Determination of toxic
equivalent of dioxins and their analogues in food
1 Scope
This Standard specifies the determination method of the contents of 17 kinds of 2,3,7,8-
substituted polychlorodibenzo-p-dioxin, polychlorino-dibenzofura (PCDD/Fs) and 12
kinds of dioxin-like polychlorinated biphenyl (DL-PCBs) and their toxic equivalent
(TEQ) in food (see Table A.1 in Appendix A).
Method I “Isotope dilution - gas chromatography - magnetic high-resolution mass
spectrometry” applies to the determination of the content of 17 kinds of PCDD/Fs and
12 kinds of DLPCBs and their TEQ in food.
Method II “Isotope dilution - gas chromatography - triple quadrupole mass
spectrometry” applies to the determination of the content of 17 kinds of PCDD/Fs and
12 kinds of DL-PCBs and its TEQ in meat and meat products, aquatic animals and their
products, milk and dairy products, eggs and egg products, oils, and fats.
Method I – Isotope dilution - gas chromatography - magnetic high-
resolution mass spectrometry
2 Principle
After the sample is extracted, purified and concentrated, measure by a gas
chromatography - magnetic high-resolution mass spectrometer and quantify by the
stable isotope dilution method; calculate the TEQ of PCDD/Fs and DL-PCBs in the
sample by accumulating the toxic equivalency factor (TEF) of each target compound
multiplied by the measured content.
3 Reagents and materials
3.1 Reagents
Unless otherwise specified, all the reagents in this method are analytical reagents, and
the water is grade-1 water specified by GB/T 6682.
3.1.1 Acetone (C3H6O): pesticide residue grade.
it and place it on a shaker to shake until the silica gel is in a uniform flow state. Prepare
it immediately before use.
3.2.3 Alkalized silica gel (33%, mass fraction): Weigh 100.0 g of active silica gel into
a 250 mL stoppered ground-rotatory flask; add 49.0 g of 1 mol/L sodium hydroxide
solution; seal it and place it on a shaker to shake until the silica gel is in a uniform flow
state. Prepare it immediately before use.
3.2.4 Silver nitrate silica gel: Weigh 10.0 g of silver nitrate into a 250 mL stoppered
ground-rotatory flask; add 40 mL of water to dissolve it; then, slowly add 90.0 g of
active silica gel; seal it and place it on a shaker to shake until the silica gel is in a uniform
flow state. Prepare it immediately before use.
3.2.5 Alkaline alumina: Take alkaline alumina and activate it at 600 °C for 24 hours.
Prepare it immediately before use.
3.2.6 Hydrous florisil (1%, mass fraction): Put an appropriate amount of florisil into a
Soxhlet extractor; use n-hexane: dichloromethane (1:1, volume ratio) to extract it for
24 hours; after evaporating, weigh 99.0 g; add 1.0 mL of water; mix evenly in a closed
container (such as a stoppered glass flask or a stoppered glass conical flask); prepare
immediately before use.
3.2.7 Mixed activated carbon: Weigh 9.0 g of activated carbon and 41.0 g of
diatomaceous earth for purification; mix evenly in a closed container (such as a
stoppered glass flask or a stoppered glass conical flask); then, activate at 130 °C for 6
hours. Prepare immediately before use.
3.2.8 Anhydrous sodium sulfate: Take anhydrous sodium sulfate and burn it at 660 °C
for 6 hours. Prepare immediately before use.
3.3 Standard solution
3.3.1 Standard solution for separation degree check: solution containing natural
PCDD/Fs. For specific compounds and concentrations, see Table B.1 in Appendix B.
3.3.2 Standard solution for PCDD/Fs isotope labeled quantitative internal standard:
solution containing 15 kinds of 13C12-PCDD/Fs. See Table B.2 for specific compounds
and concentrations.
3.3.3 Standard solution for PCDD/Fs isotope labeled recovery rate internal standard:
solution containing 13C12-1,2,3,4-TCDD and 13C12-1,2,3,7,8,9-HxCDD. See Table B.3
for specific concentrations.
3.3.4 Standard solution for PCDD/Fs calibration: standard solution containing natural
and isotope-labeled PCDD/Fs series calibration curve. See Table B.4 for specific
compounds and concentrations.
3.3.5 Standard solution for DL-PCBs isotope labeled quantitative internal standard:
solution containing 12 kinds of 13C12-DL-PCBs. See Table B.5 for specific compounds
and concentrations.
3.3.6 Standard solution for DL-PCBs isotope labeled recovery rate internal standard:
solution containing 13C12-PCB 70, 13C12-PCB 111 and 13C12-PCB 170. See Table B.6
for specific concentrations.
3.3.7 Standard solution for DL-PCBs calibration: standard solution containing natural
and isotope-labeled DL-PCBs series calibration curve. See Table B.7 for specific
compounds and concentrations.
3.3.8 Standard solution for sensitivity check: Standard solution containing natural and
isotope-labeled PCDD/Fs series. See Table B.8 for specific compounds and
concentrations.
Note: Store the standard solution at room temperature in the dark, where it has a shelf
life of 6 years.
4 Instruments and apparatuses
4.1 Gas chromatography - magnetic high-resolution mass spectrometer (GC-HRMS)
4.2 Balance: The sensitivity is 0.1 g and 0.001 g.
4.3 Tissue homogenizer.
4.4 Pulverizer.
4.5 Freeze dryer.
4.6 Rotary evaporator.
4.7 Nitrogen concentrator.
4.8 Ultrasonic cleaner.
4.9 Shaker.
4.10 Soxhlet extractor, equipped with extraction sleeve.
4.11 Accelerated solvent extraction instrument (optional).
4.12 Muffle furnace: able to maintain constant temperature (±10 ℃) within the range
of 200 ℃ ~ 700 ℃.
4.13 Oven: able to maintain constant temperature (±5 ℃) within the range of 105 ℃ ~
250 ℃.
4.14 Glass chromatography column: equipped with polytetrafluoroethylene plunger,
150 mm (length) × 8 mm (inner diameter), 300 mm (length) × 15 mm (inner diameter).
4.15 Fully automatic sample purification system: equipped with acid-base composite
silica gel column, alkaline alumina column and activated carbon purification column.
4.16 Gel permeation chromatography column (GPC): glass column (inner diameter 15
mm ~ 20 mm), containing 50 g of S-X3 gel, or equivalent fully automatic GPC.
5 Analysis steps
5.1 Sample preparation
5.1.1 General food samples: After dry food is crushed evenly, weigh 10 g ~ 20 g
(accurate to 0.01 g) for later use; for milk powder, weigh 15 g (accurate to 0.01 g) for
later use; after other samples are homogenized, weigh 50 g ~200 g (accurate to 0.01 g);
freeze-dried for later use.
5.1.2 Grease sample: directly weigh 5 g of sample (accurate to 0.01 g) into an eggplant-
shaped bottle; add 10 μL each of standard solutions for PCDD/Fs and DL-PCBs isotope
labeled quantitative internal standard; wait for purification.
5.2 Extraction
5.2.1 Soxhlet extraction
Before extraction, put an empty extraction sleeve into the Soxhlet extractor (4.10); use
n-hexane: methylene chloride (1:1, volume ratio) as the extraction solvent; pre-extract
at a reflux speed of 3 times/h ~ 4 times/ h for 8 hours; then, take it out to dry.
Place the sample (5.1.1) in a ceramic mortar; add anhydrous sodium sulfate and grind
it to make a free-flowing powder. Transfer to a pre-cleaned sleeve; respectively add 10
μL each of standard solutions for PCDD/Fs and DL-PCBs isotope labeled quantitative
internal standard; use glass wool to cover the sample; balance for 30 minutes and put it
into a Soxhlet extractor; use n-hexane: dichloromethane (1:1, volume ratio) as the
extraction solvent; carry out extraction for 18 h ~ 24 h at a reflux rate of 3 times/h ~ 4
times/h.
5.2.2 Accelerated solvent extraction
Place the sample (5.1.1) in a ceramic mortar; grind it and mix it evenly with an
appropriate amount of diatomaceous earth; transfer it to the extraction tank; add 10 μL
each of standard solutions for PCDD/Fs and DL-PCBs isotope labeled quantitative
internal standard. After sealing, place it on an accelerated solvent extraction instrument
for extraction. The extraction reference conditions are: extraction solvent: n-hexane:
dichloromethane (1:1, volume ratio); pressure: 10.3 MPa; temperature: 150 °C; static
extraction time: 10 min; cycle: 1 time.
Depending on laboratory conditions, choose to use acidified silica gel for fat removal
and/or gel permeation chromatography for fat removal.
5.4 Purification and separation
5.4.1 Packed column for purification
5.4.1.1 Composite silica column for purification
Take a glass chromatography column with an inner diameter of 15 mm; fill the bottom
with an appropriate amount of glass wool; sequentially add 2 g of active silica gel, 5 g
of alkalized silica gel, 2 g of active silica gel, 10 g of acidified silica gel, 2 g of active
silica gel, 5 g of silver nitrate silica gel, 2 g of active silica gel and 2 g of anhydrous
sodium sulfate; pack by the dry method; tap the chromatography column to make it
evenly distributed.
First use 150 mL of n-hexane to pre-elute. When the liquid level drops to about 2 mm
above the anhydrous sodium sulfate layer, close the column valve and discard the eluent.
Check the chromatography column and repack it if channeling occurs. Add the
degreased and concentrated extract; use 5 mL of n-hexane to wash the eggplant-shaped
bottle (5.3) twice; add them to the column together; open the column valve; when the
liquid level drops to the anhydrous sodium sulfate layer, add 400 mL of n-hexane; elute
at a flow rate of 1 drop/s ~ 2 drops/s; use the eggplant-shaped bottle to collect the eluate;
use a rotary evaporator to concentrate to 3 mL ~ 5 mL, for the next step of purification.
Avoid bumping during concentration.
5.4.1.2 Alkaline alumina column for separation
Take a glass chromatography column with an inner diameter of 15 mm; fill the bottom
with an appropriate amount of glass wool; then add 25 g of alkaline alumina and 10 g
of anhydrous sodium sulfate in sequence. Pack by the dry method; tap the column
lightly to distribute it evenly. First use 150 mL of n-hexane to pre-elute. When the liquid
level drops to about 2 mm above the alumina, close the column valve. Discard the eluent.
Check the chromatography column and refill the column if channeling occurs. Add the
extract purified by the mixed silica gel column; use 5 mL of n-hexane to wash the
eggplant-shaped bottle (5.4.1.1) twice; add them to the column together. Use 60 mL of
n-hexane to elute the alumina column; discard the eluent. Use 90 mL of toluene to elute
at a flow rate of 1 mL/min ~ 2 mL/min; use an eggplant-shaped bottle to collect the
eluate for DL-PCBs analysis. Then, use 200 mL of n-hexane: methylene chloride (1:1,
volume ratio) to elute at a flow rate of 1 drop/s ~ 2 drops/s; use an eggplant-shaped
bottle to collect the eluent for PCDD/Fs analysis. For each eluent, concentrate to 1 mL
~ 2 mL using a rotary evaporator; then, add 50 mL of n-hexane; concentrate to 1 mL ~
2 mL. Avoid bumping during concentration.
5.4.1.3 Alkaline alumina column for purification
Further purification of the eluent containing PCDD/Fs components: Take a glass
chromatography column with an inner diameter of 8 mm; fill the bottom with an
appropriate amount of glass wool; then add 2.5 g of alkaline alumina and 2 g of
anhydrous sodium sulfate in sequence. Pack by the dry method; tap the column lightly
to distribute it evenly. Use 20 mL of n-hexane to pre-elute; discard the eluent. Add the
concentrated eluate. Use 40 mL of n-hexane: methylene chloride (98:2, volume ratio)
to eluent; discard the eluent. Use 30 mL of n-hexane: dichloromethane (1:1, volume
ratio) to elute at a flow rate of 1 drop/s ~ 2 drops/s; use an eggplant-shaped bottle to
collect the eluate; use a rotary evaporator to concentrate to 1 mL ~ 2 mL. Avoid bumping
during concentration.
Further purification of the eluent containing DL-PCBs components: Take a glass
chromatography column with an inner diameter of 8 mm; fill the bottom with an
appropriate amount of glass wool; then add 2.5 g of alkaline alumina and 2 g of
anhydrous sodium sulfate in sequence. Pack by the dry method; tap the column lightly
to distribute it evenly. Use 30 mL of n-hexane: methylene chloride (99:1, volume ratio)
to pre-eluent; discard the eluent. Add the concentrated eluent; use 15 mL of n-hexane:
dichloromethane (1:1, volume ratio) to elute at a flow rate of 1 drop/s ~ 2 drops/s; use
an eggplant-shaped bottle to collect the eluate; use a rotary evaporator to concentrate to
1 mL ~ 2 mL. Avoid bumping during concentration.
5.4.2 Fully automatic sample purification system for purification
Connect the acid-base composite silica gel column, alkaline alumina column and
activated carbon purification column to the fully automatic sample purification system
in sequence; prepare each elution solution according to the program and connect the
pipelines (see Figure D.1). Transfer the degreased and concentrated extract to the
injection tube of the fully automatic sample purification system. Elute sequentially
according to the elution program (see Table D.1); purify and separate the sample; use
the eggplant-shaped bottle to collect the eluate containing PCDD/Fs and DL-PCBs
components respectively; use a rotary evaporator to concentrate to 1 mL ~ 2 mL; then,
add 50 mL of n-hexane and concentrate to 1 mL ~ 2 mL. Avoid bumping during
concentration.
According to laboratory conditions, choose to use packed column for purification or
fully automatic sample purification system for purification. If the PCDD/Fs purification
effect cannot meet the measurement requirements, choose activated carbon column
and/or florisil column for further purification.
5.4.3 Supplementary purification methods
5.4.3.1 Activated carbon column for purification
Take a 10 mL disposable glass pipette; cut off both ends; make a glass tube about 10
cm long; put in an appropriate amount of glass wool and plug it tightly; then, add 0.55
g of mixed activated carbon; then, put in an appropriate amount of glass wool; compact
5.7.1.1 Gas chromatography reference conditions
The gas chromatography reference conditions are as follows:
a) Chromatographic column: 5% diphenyl-95% dimethylpolysiloxane column, 60
m × 0.25 mm × 0.25 μm, or equivalent;
b) Temperature of the sample injector: 280 ℃;
c) Injection mode: splitless injection, constant flow mode;
d) Injection volume: 2 μL;
e) Transmission line temperature: 310 ℃;
f) Column temperature: 120 ℃ (maintain for 1 min); rise to 220 ℃ at 43 ℃/min
(maintain for 15 min); rise to 250 ℃ at 2.3 ℃/min, to 260 ℃ at 0.9 ℃/min, and
to 310 ℃ at 20 ℃/min (maintain for 9 min);
g) Carrier gas: high purity helium ( >99.999%), 0.8 mL/min.
5.7.1.2 Mass spectrometry reference conditions
The mass spectrometry reference conditions are as follows:
a) Ionization mode: electron impact source (EI);
b) Electron energy: 45 eV;
c) Reference gas: perfluorotributylamine (FC-43) or perfluorinated kerosene (PFK);
d) Resolution: ≥10 000, reference ion: 313.983 3 (FC-43) or 342.978 7 (PFK);
e) Ion source temperature: 270 ℃;
f) Ion monitoring mode: multiple ion monitoring (MID);
g) Monitoring ions: See Table C.1 for the accurate mass of monitoring ions for each
compound.
5.7.2 DL-PCBs analysis conditions
5.7.2.1 Gas chromatographic conditions
The gas chromatographic conditions are shown as below:
a) Chromatographic column: Same as a) in 5.7.1.1;
b) Temperature of the sample injector: 290 ℃;
Under given conditions, separately inject the CS1 standard solution for PCDD/Fs
calibration standard solution and DL-PCBs calibration standard solution. The relative
retention time of each target compound shall comply with the provisions of Table C.2.
5.8.4 Ion abundance ratio
Under given conditions, separately inject the CS1 standard solution for PCDD/Fs
calibration standard solution and DL-PCBs calibration standard solution. The ion
abundance ratio of each target compound shall comply with the provisions of Table C.3.
5.9 Preparation of the standard curve
5.9.1 Relative response factor
Inject the PCDD/Fs calibration standard solution and DL-PCBs calibration standard
solution into GC-HRMS in order of concentration from low to high, and obtain the peak
area. Calculate the relative response factor (RRF) of each target compound according
to Formula (3). Formula (3) is applicable to other PCDD/Fs and DL-PCBs than
1,2,3,7,8,9-HxCDD and OCDF.
Where:
An1 and An2 – peak areas of the first and second mass ions of the target compound;
cl – concentration of quantitative internal standard, in nanograms per milliliter (ng/mL);
Al1 and Al2 – peak areas of the first and second mass ions of the quantitative internal
standard;
cn – concentration of the target compound, in nanograms per milliliter (ng/mL).
Within the concentration range of the calibration standard solution, the relative standard
deviation of the RRF of each compound is ≤20%.
5.9.2 Response factor
5.9.2.1 Response factors of 1,2,3,7,8,9-HxCDD and OCDF
Inject the PCDD/Fs calibration standard solution into the GC-HRMS in order from low
to high concentration to obtain the peak area. Calculate the response factors (RF) of
1,2,3,7,8,9-HxCDD and OCDF according to Formula (4) (for 1,2,3,7,8,9-HxCDD, use
13C12-1,2,3, 6,7,8-Hx-CDD as the quantitative internal standard; for OCDF, use 13C12-
OCDD as the quantitative internal standard).
Report the results in terms of fat mass and convert according to the fat content (5.2.3)
in the sample.
7 Precision
Intra-laboratory repeatability, calculated as TEQtotal, RSD≤15%.
8 Detection-limit
Based on a sampling volume of 50 g, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-
TCDD) and 2,3,7,8-tetrachloro-dibenzofura (2,3,7,8-TCDF) are 0.04 ng/kg,
octachlorodibenzo-p-dioxin (OCDD) and octachloro-dibenzofuran (OCDF) are 0.40
ng/kg, the remaining PCDD/Fs are 0.20 ng/kg, and DL-PCB is 1.00 ng/kg.
9 Others
9.1 After the organic reagents used for analysis are concentrated 10 000 times,
PCDD/Fs and DL-PCBs with S/N > 3 shall not be detected.
9.2 The S/N of 2,3,7,8-TCDD in the blank test shall not be greater than 3.
9.3 The laboratory used to detect dioxins and their analogs in food shall be set up
separately; the laboratory shall not be shared with other testing items, especially the
detection of dioxins and their analogs in environmental samples. During the detection
process, standard solutions, sample extraction solutions and sample extraction residues
added with stable isotopes to replace the standards shall be used and stored with caution
to avoid laboratory contamination. During the detection process, experimenters shall
wear gloves, adsorbent masks, safety glasses, and lab coats to avoid unnecessary
personal contact and exposure. The laboratory shall be equipped with a dedicated
independent laboratory ventilation system with good performance; the fume hood shall
meet the requirements for negative pressure operation. An activated carbon adsorption
device shall be installed at the end of its exhaust outlet, and the adsorption material shall
be replaced regularly to ensure that exhaust gas emissions meet environmental
protection requirements. Liquid and solid waste generated during sample extraction,
purification, concentration and analysis in laboratories of dioxins and their analogues
shall be collected and disposed of in accordance with national and local waste
management regulations.
Method II – Isotope dilution - gas chromatography - triple
quadrupole mass spectrometry
13.7 Apparatus reference conditions
13.7.1 PCDD/Fs analysis conditions
13.7.1.1 Gas chromatography reference conditions
Same as 5.7.1.1.
13.7.1.2 Mass spectrometry reference conditions
The mass spectrometry reference conditions are as follows:
a) Resolution: The resolution of the quadrupole shall be better than or equal to the
unit mass resolution.
b) Ionization mode: electron impact source (EI), energy 70 eV; or atmospheric
pressure chemical ionization source (APCI), corona needle current 3 μA.
c) Ion source temperature: 280 °C for electron impact source (EI); or 150 °C for
atmospheric pressure chemical ionization source (APCI).
d) Data acquisition mode: Multiple reaction ion monitoring mode (MRM), which
monitors two specific parent ions and one product ion generated by each parent
ion for all compounds. The specific information on the ion pairs of each
compound is shown in Table C.4.
13.7.2 DL-PCBs analysis conditions
13.7.2.1 Gas chromatography reference conditions
Chromatographic column: Same as 5.7.2.1.
13.7.2.2 Mass spectrometry reference conditions
Same as 13.7.1.2.
13.8 Instrument performance requirements
13.8.1 Resolution
Same as 5.8.1.
13.8.2 Sensitivity
Inject the sensitivity check standard solution, the deviation between the RRF (RF) of
the target compound and the average RRF (RF) of the target compound calculated by
the calibration standard curve is less than 30%.
13.8.3 Ion abundance ratio
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.