Determination of dioxins and dioxin - like PCBs (DL - PCBs) in food Mr C.S. NG, Dr P.W. KONG The Government Laboratory of HKSAR
Determination of dioxins and dioxin-like PCBs (DL-PCBs) in food
Mr C.S. NG, Dr P.W. KONGThe Government Laboratory of HKSAR
Part I: Background information
Mr C.S. NGThe Government Laboratory of HKSAR
(3) Polychlorinated Biphenyls (PCBs)
(1) Polychlorinated Dibenzo-p-dioxins
(2) Polychlorinated Dibenzofurans
O
OClx
Cly
O Cly
Clx
Cly
Clx
A Group of Chlorinated Organic Compounds
Dioxins refer to Polychlorinated Dibenzo-p-dioxins (PCDD, 75 isomers) and Polychlorinated Dibenzofurans (PCDF, 135 isomers)
PCBs refers to Polychlorinated Biphenyls (PCB, 209 isomers)
What is Dioxins and PCBs
Occurrence• Air
– ambient air, stack gas, occupational• Water
– marine water, river water, rain• Soil & Sediment• Both Dioxins and PCBs do not readily degrade.
They persist in the environment and tend to bioaccumulate up the food chain, bringing widespread environmental occurrence.
• Dioxins and PCBs share a number of common behaviour such as fat solubility and their persistence in the environment, however, their level in the environment and in food are vary.
Analysis of Dioxins and PCBs
• All 75 PCDD, 135 PCDF and 209 PCB congeners?
One of the most harmfuldioxins is known as TCDD(2,3,7,8-tetrachlorodibenzo-
p-dioxin)
+ 16 other 2,3,7,8-
substituted congenersPCDD PCDF
O
OClx
ClyO Cly
Clx1
4
3
29
8
7
6
1
4
3
29
8
7
6
Dioxins (17) and “Dioxin-like” PCBs (12)
Cly
Clx
PCB
ortho
ortho
PCB 77*PCB 81*PCB 126*PCB 169*PCB 105PCB 114
PCB 118PCB 123PCB 156PCB 157PCB 167PCB 189
4 non-ortho* and 8 mono-ortho “dioxin-like” PCBs
• 2,3,7,8-TCDD has been designated carcinogenic by the US EPA and IARC (International Agency for Research on Cancer)
• Among 210 PCDD and PCDF congeners, 17 individual 2,3,7,8-chlorine substituted congeners is ranked in relation to the toxicity of 2,3,7,8-TCDD.
• Among 209 PCB congeners, 4 non-ortho and 8 mono-ortho chlorine substituted congenersare also selected for toxicity consideration.
• Numerical Toxic Equivalent Factors (TEFs) have been developed– The toxicity of different PCDDs, PCDFs and PCBs are compared with
the toxicity of TCDD and a TEF is assigned relative to TCDD, where TCDD is assigned to has TEF of 1
All isomers ?
Compound 1998 TEF 2005 TEF
2,3,7,8-TCDD 1 1
1,2,3,7,8-PeCDD 1 1
1,2,3,4,7,8-HxCDD 0.1 0.1
1,2,3,6,7,8-HxCDD 0.1 0.1
1,2,3,7,8,9-HxCDD 0.1 0.1
1,2,3,4,6,7,8-HpCDD 0.01 0.01
OCDD 0.0001 0.0003
2,3,7,8-TCDF 0.1 0.1
1,2,3,7,8-PeCDF 0.05 0.03
2,3,4,7,8-PeCDF 0.5 0.3
1,2,3,4,7,8-HxCDF 0.1 0.1
1,2,3,6,7,8-HxCDF 0.1 0.1
1,2,3,7,8,9-HxCDF 0.1 0.1
2,3,4,6,7,8-HxCDF 0.1 0.1
1,2,3,4,6,7,8-HpCDF 0.01 0.01
1,2,3,4,7,8,9-HpCDF 0.01 0.01
OCDF 0.0001 0.0003
Compound 1998 TEF 2005 TEF
3,3′,4,4′-tetraCB (PCB 77) 0.0001 0.0001
3,4,4′,5-tetraCB (PCB 81) 0.0001 0.0003
3,3′,4,4′,5-pentaCB (PCB 126) 0.1 0.1
3,3′,4,4′,5,5′-hexaCB (PCB 169) 0.01 0.03
2,3,3′,4,4′-pentaCB (PCB 105) 0.0001 0.00003
2,3,4,4′,5-pentaCB (PCB 114) 0.0005 0.00003
2,3′,4,4′,5-pentaCB (PCB 118) 0.0001 0.00003
2′,3,4,4′,5-pentaCB (PCB 123) 0.0001 0.00003
2,3,3′,4,4′,5-hexaCB (PCB 156) 0.0005 0.00003
2,3,3′,4,4′,5′-hexaCB (PCB 157) 0.0005 0.00003
2,3′,4,4′,5,5′-hexaCB (PCB 167) 0.00001 0.00003
2,3,3′,4,4′,5,5′-heptaCB (PCB 189) 0.0001 0.00003
Toxicity Equivalent Factor(TEF)
• Sum of product of concentration ofindividual isomer and corresponding TEF
• TEQ = PCDDn(TEF)n + PCDFn(TEF)n+ PCBn(TEF)n
Toxicity Equivalent (TEQ)
Part II: Determination of dioxins and dioxin-like PCBs by Gas Chromatography –High Resolution Mass Spectrometry
Dr P.W. KONGThe Government Laboratory of HKSAR
EU regulation Commission Regulation (EU) 2017/644 of 5 April 2017 laying down
methods of sampling and analysis for the control of levels of dioxins, dioxin-like PCBs and non-dioxin-like PCBs in certain foodstuffs
Commission Regulation (EU) 2017/644 of 5 April 2017 laying down methods of sampling and analysis for the control of levels of dioxins, dioxin-like PCBs and non-dioxin-like PCBs in certain foodstuffs GC-HRMS
GC-MS/MS
Bioanalytical screening method (e.g. cell-based assays, receptor-assays or immunoassays)
(since commission regulation (EU) 589/2014)
Confirmatory methods
EU regulation Commission Regulation EU 1259/2011 setting maximum levels of
dioxins, dioxin-like PCBs non-dioxin-like PCBs in foodstuffs
Maximum levels for sum of TEQ for different foodstuffs based on wet weight or fat basis
International Standard Methods
Anal Bioanal Chem (2006) 386:791-806
GL Method
Dioxins Dioxins-like PCBs
GL method is referenced to EPA method 1613 and 1668, for dioxins and dioxin-like PCBs, respectively
Isotope dilution techniques with 13C-labelled standards
Instrumentation: GC-HRMS
Sample Preparation Workflow
Sample (~ 40 g)+ labelled IS+ blending
Homogenization& extraction
Acidic SilicaChromatography
Fractionation bycarbon column
Alumina columnPre-concentration
+ injection IS
GC-HRMS
extraction
Clean-up
Pre-concentration
GC-HRMS
1
2
3
4
Labelled IS added
Injection IS added
Clean-upAcidic silica gel chromatography
Fat is one of the major composition of food.
The acidic silica gel can hydrolyze the fat.
This is used to remove fat/lipids in sample.
Purpose
Clean-upCarbon Column
Dioxins, dioxin-like PCBs and PBDE in the concentrated extract are fractionated by carbon column chromatography into different fractions according to their interactions with carbon (PX-21 carbon).
To separate different contaminants, which often exist at different order of magnitude in food.
Gradient elution with hexane, DCM, ethyl acetate, toluene for PCBs followed by reversed elution with toluene for dioxins
Purpose
F2 PCB
F3 Dioxin
F1 PBDE
Automated clean-up system
Disposable carbon column
Sample extract
F3 Dioxin
Clean-upAlumina column
Each fraction (dioxin faction and dioxin-like PCB faction) collected after carbon column fractionation was further process by alumina mini-column. The fractions are then concentrated to a final volume of 20 µL before GC-HRMS analysis.
Purpose
20 mL
Confirmatory Analysis by GC-HRMS Two sequences were analysed (dioxin fraction, and PCB
fraction)
Isotope Dilution Method
Samples were spiked with injection internal standard prior to HRMS analysis
Quantitation by SIM mode and identification of congeners through ion abundance ratio between 2 monitored ions
Advantages of HRMS
exact masses are measured, instead of nominal masses
All with same nominal mass of 28
Low resolving power
High resolving power
Ability to distinguish two peaks of similar m/z
Advantage of HRMS
identification with high confidence
eliminate potential interference
High Accuracy
High resolution
Confirmatory Analysis by GC-HRMS GC column: DB-5MS 5% Phenyl Methyl Siloxane, 60 m, 0.25 mm id, 0.25
um film
If some 2,3,7,8-substituted isomers of dioxins were identified on DB-5MS, alternative GC column should be performed.
If the total TEQ of the sample found to be higher than maximum level set in commission regulation (EU) 1259/2011, alternative GC columns for both dioxins and PCBs should be performed.
Dioxins: Supelco SP-2331, 60 m, 0.25 mm id, 0.2 um filmPCBs: SGE HT-8, 50 m, 0.22 nn id, 0.25 um film
Calibration Two calibration: dioxin (CS1-CS5) and PCBs (CS1-CS7)
The calibration curve is used to calculate the relative response factor for each congener of interest
An = sum of area response of primary and secondary m/z (M and M+2) for native congenerAl = sum of area response of primary and secondary m/z (M and M+2) for labelled congener
Cl = conc. of labelled congener
Cn = conc. of native congener
The average relative response factors are used to quantify the native congeners in samples by the isotope dilution method
RR = Relative response
Calibration
Calibration
System Performance Criteria - HRMS Mass spectrometer (MS) resolution (R) ≥ 10,000
10 % peak height
m : mass number of the observed massΔm : the mass difference between two adjacent peaks that are just resolved
In magnetic sector mass spectrometers, peaks are usually defined to be separated down to a 10% valley
System Performance Criteria - HRMS Mass-drift correction is mandatory and a lock-mass using PFK is used for
correction.
System Performance Criteria –GC resolution 2,3,7,8-TCDD and other tetra-dioxin isomers, and 2,3,7,8-TCDF and the
other tetra-furan isomers are resolved with a valley of 25%
25%
25%
2,3,7,8-TCDD
2,3,7,8-TCDF
Reference materials
Reference materials
Analytes Description Uses
PCDDs/PCDFs EDF-9999 Method 1613 Calibration solutions (with 13C-labelled IS) CS1-CS5
Calibration
EDF-7999 Method 1613 Precision and Recovery Standard Solution
Spike samples
EDF-8999 Method 1613 Labelled Compound Stock Solution
Labelled internal standards
EDF-5999 Method 1613 Internal Standard Spiking Solution
Injection internal standards
EDF-4147 Window Definer and Isomer Specificity Mix Window defining solution and isomer specificity
Reference materials
Analytes Description Uses
PCBs WP-CVS Dioxin-Like PCBs Calibration and Verification Solutions CS1-CS7
Calibration
WP-STK Native PCB Solution Spike samples
WP-LCS Surrogate Spiking Solution Labelled internal standard
WP-ISS Internal Standard Solution Injection internal standard
Proficiency Test Programme on Dioxins
Programme Starting timeNorwegian Institute of Public Health -Interlaboratory Comparision of Persistent Organic Pollutants in food (ILC POPs)
Spring (every year)
Bipea – PCB & PAH in food several rounds spanning from October to May
EU-RL Proficiency Test on Determination of PCDD/Fs and PCBs in Feed of Plant Origin
July (every year)
The EndThank you for your attention !
This presentation is for your reference only.