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

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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.