lnnovative strategy for Dioxins/PCBs extraction and ...Vincent Vaccher| RAFA 2015| 04-11-2015| PRAGUE 4/32 1. LABERCA in few slides... •Research Department of Oniris (Nantes-Atlantic

Vincent Vaccher| RAFA 2015| 04-11-2015| PRAGUE 1/32

Laboratoire d’Étude des Résidus et Contaminants dans les Aliments (LABERCA)USC INRA 1329, Oniris, LUNAM Université

BP 50707, 44307 Nantes Cedex 3, France - www.laberca.org

Vincent Vaccher, Philippe Marchand, Claudia BlumElodie Lesquin, Danielle Piquet, Anaïs Venisseau

lnnovative strategy for

Dioxins/PCBs extraction and purification

RAFA 2015 – 4th November 2015, Prague


Extraction : SpeedExtractor(BUCHI)

Purification : Autosym GO-4-HT(MIURA -SHIMADZU)

+

Evaluate the coupling

Objective of this study


Outline

1. LABERCA in a few slides...

2. Reminders about Dioxins/PCBs and the analysis method

3. Extraction of Dioxins and PCBs

4. Purification of Dioxins and PCBs

5. Method performances

6. Conclusions : advantages/drawbacks


1. LABERCA in few slides...

• Research Department of Oniris (Nantes-Atlantic National College of Veterinary Medicine,

Food Science and Engineering) based in Nantes (France)

• Laboratory of 60 persons dedicated to the chemical safety of food

• Research activites on the chemical safety of food (projects at regional, national, European and

international level)

• French National Reference Laboratory for dioxins, DL-PCBs, NDL-PCBs, PAHs, melamine and growth promoters

Methods are accreditedOrganisation of PT is accredited

• Teaching (students and international training)


HPLC (x 2) – CHIP (x1) GC-MS (x 3)

GC-MS/MS (x 2) LC-MS/MS (x 2)

GC-HRMS (x 3) GC-C-IRMS (x 2)

LC-HRMSn (x 2)

LC-MS3 (x 1)

1. LABERCA in a few slides...


Dioxins

Toxic substancesReleased in the environment (incomplete combustion processes)Cancerogenic

PCDDs PCDFs

Produced in large volumes for industrial applications

PCBs

- 12 PCBs classified as “dioxin-like” (DL PCBs)

- 6 PCBs defined as “non-dioxin like” PCBs (NDL PCBs),

Lipophilic and accumulated in human and animal tissue

2. Reminders: Dioxins and PCBs

209 congeners :

Extraction of sample fat with SpeedExtractor

https://de.wikipedia.org/wiki/Datei:PCDD_general_structure.svg

https://de.wikipedia.org/wiki/Datei:PCDD_general_structure.svg

http://www.google.ch/imgres?imgurl=https://upload.wikimedia.org/wikipedia/commons/thumb/6/64/PCB_structure_general.svg/220px-PCB_structure_general.svg.png&imgrefurl=https://de.wikipedia.org/wiki/Polychlorierte_Biphenyle&h=69&w=220&tbnid=Vav_X38MkNxEJM:&docid=CiKLjTN_pWwWvM&ei=k7blVYbPN8W3sQGQjZ3QCw&tbm=isch&ved=0CHsQMyhUMFRqFQoTCMbkqamG1scCFcVbLAodkEYHug

http://www.google.ch/imgres?imgurl=https://upload.wikimedia.org/wikipedia/commons/thumb/6/64/PCB_structure_general.svg/220px-PCB_structure_general.svg.png&imgrefurl=https://de.wikipedia.org/wiki/Polychlorierte_Biphenyle&h=69&w=220&tbnid=Vav_X38MkNxEJM:&docid=CiKLjTN_pWwWvM&ei=k7blVYbPN8W3sQGQjZ3QCw&tbm=isch&ved=0CHsQMyhUMFRqFQoTCMbkqamG1scCFcVbLAodkEYHug


Preparation of sample(freeze-dried or only dried)

Extraction SpeedExtractor E-914

PurificationMIURA GO-4 HT

Identification and quantification GC-HRMS

2. Reminders: principle of the analysis method


Preparation of sample(freeze-dried or only dried)

Extraction SpeedExtractor E-914

PurificationMIURA GO-4 HT

Identification and quantification GC-HRMS

2. Reminders: principle of the analysis method


SpeedExtractor E-914



gaseous

Principle of the Pressurized Liquid Extraction (PLE)

liquid

Temperature T

Pre

ssure

p

p1

T1 T2

liquid

gaseous

p2

solid

Elevatep

ElevateT



SpeedExtractor E-914

• Higher analyte solubility at high temperatures

- Increased capacity of solvents to solubilize analytes

- Faster diffusion rates

• Better penetration of solvent in the sample

at high temp./press

- Disruption of strong solute-matrix interactions

- Decreased viscosity of organic solvents

Elevated temp./press high speed less solvent consumption



1st innovation: parallel approach : 4 or 6 samples in 20 min


- Solvents- Volume of solvent- Temperature- The number of cycles

Differents parameterscan be adapted :


N2

0

30

45 15

Flush with N2

Flush into vial

Discharge

Hold (extract)

Heat up (load)

Preheat

Flush into waste



Flush the lines

N2

StartFlush with N2

Flush into vial

Discharge

Hold (extract)

Heat up (load)

Preheat

Flush into waste

0

30

45 15



Heat up – fill the cell, apply pressure, hold (static time)

N2

30

45 15

Analyte

Matrix

Flush with N2

Flush into vial

Discharge

Hold (extract)

Heat up (load)

Preheat

Flush into waste

0



Discharge – Remove the extracts

N2

30

45 15

Matrix

Analyte

Flush with N2

Flush into vial

Discharge

Hold (extract)

Heat up (load)

Preheat

Flush into waste

0



2nd innovation: nitrogen ONLY USED for purging system(very low consumption)

N2

30

45 15

Flush with N2

Flush into vial

Discharge

Hold (extract)

Heat up (load)

Preheat

Flush into waste

Matrix

Analyte

0



3 heater blocks

1st module 2nd module …..


3rd innovation: heater blocks – better interaction



• 4 columns, individually packed

4th innovation: disposable materials (low cross contamination)


GO-4-HT - Miura

Silver nitrate silica gel 10% (w/w)

Sulfuric acid silica gel44% (w/w)

Carbon column

Alumina column



85-90 mL Hexane

Sample extract

A- 0.8-1 mL Toluene

A: Mono/di-orthoPCBs

B- 0.8-1 mL TolueneB: Non-ortho PCBs

and PCDD/Fs

Waste


60 °C

90 °C

5th innovation:very low solvent volume


• Selection of matrices

- From proficiency tests (EURL Dioxins/PCBs, Norwegian Institute of Public Health)

- Different kinds of matrices

- Different levels of concentration

- Known to cause « sometimes » extraction problems

Egg powder ≥ MRL

57% of fat

Milk powder ≤ MRL

20% of fat

Pork meat < MRL

26% of fat

Herring << MRL

6 % of fat

NDL-PCBs concentrations level

5. Results

Milk fat ≥ MRL

98% of fat


• Isotopic dilution quantification with a GC-HRMS

• Results were expressed according to European regulationrequirements (589/2014 and 1259/2011)

• Each matrix analysed 4 times

5. Results


RSD (%)

Milk powder Milk fat Egg powder Herring Pork

Fat content [%] 3.8 0.3 0.6 0.6 1.9

• Repeatability (n=4)

Excellent repeatabilityMost of them < 5 %

TEQ PCDD/Fs 5.7 10.2 10.2 10.2 4.6

TEQ DL-PCBs 4.8 0.6 2.7 0.6 4.8

TEQ TOTAL 3.1 5.1 2.5 0.7 4.5

Sum of NDL-PCBs 3.6 1.8 0.6 0.1 6.5

5. Results


TEQ

WH

O 2

00

5 P

CD

D/F

TEQ

WH

O 2

00

5 P

CB

-DL

TEQ

WH

O 2

00

5 P

CD

DF

+PC

B-D

L

Satisfactory accuracy- for Dioxins, DL-PCBs and NDL-PCBs- for all matrices analysed

• Accuracy

5. Results


Z-SCORES

σ (%) Milk powder Milk fat Egg powder Herring Pork

25 Fat content -0,2 - 0,1 -0,9 -0,3

• Accuracy

Satisfactory recalculated Z-scores

15 TEQ PCDD/Fs 0,5 1,1 -1,3 -1,2 0

15 TEQ DL-PCBs -0,1 -0,1 0,1 -0,8 1,7

15 TEQ TOTAL 0 -0,6 -0,7 -1,0 0,1

10 Sum of NDL-PCBs -0,1 -0,5 -0,4 -1,2 2,9

5. Results


Milk powder : extraction problem due to granulomety

Accurate for all dioxins and PCBs congeners

5. Results


Compound View JEOL DioK V4.02 2013/08/01 14:31:39 Page 1

DqData: Diox2-020712 MIURA (EPA-1613, DB-5MS), Injection= blanc 2 T1 (UNK)

Original: Diox2-020712001.mfl, InjectionNo= 60, Sample= Blanc 2 T1, Date= <Date>

37.2 37.3 37.4 37.5 37.6 37.7 37.8 37.9 38.0 38.1

Retention Time (min)

(1863)

(3547)

0

20

40

60

80

100

Inte

nsity

O8CDD / Average

37.2 37.3 37.4 37.5 37.6 37.7 37.8 37.9 38.0 38.1


(442672)

0

20

40

60

80

100

Inte

nsity

13C-O8CDD / Average

37.2 37.3 37.4 37.5 37.6 37.7 37.8 37.9 38.0 38.1


(1385)

(2867)

0

20

40

60

80

100

Inte

nsity

O8CDF / Average

37.2 37.3 37.4 37.5 37.6 37.7 37.8 37.9 38.0 38.1


(519710)

0

20

40

60

80

100In

ten

sity

13C-O8CDF / Average

Compound View JEOL DioK V4.02 2013/08/01 14:29:03 Page 1

DqData: Diox2-290713b (EPA-1613, DB-5MS), Injection= blanc ase1 (UNK)

Original: Diox2-290713001.mfl, InjectionNo= 38, Sample= blanc3, Date= <Date>

37.4 37.5 37.6 37.7 37.8 37.9 38.0 38.1 38.2 38.3 38.4


(2089)

(3757)

0

20

40

60

80

100

Inte

nsity

O8CDD / Average

37.4 37.5 37.6 37.7 37.8 37.9 38.0 38.1 38.2 38.3 38.4


(111795)

0

20

40

60

80

100

Inte

nsity

13C-O8CDD / Average

37.5 37.6 37.7 37.8 37.9 38.0 38.1 38.2 38.3 38.4 38.5 38.6


(1478)

(2419)

0

20

40

60

80

100

Inte

nsity

O8CDF / Average

37.5 37.6 37.7 37.8 37.9 38.0 38.1 38.2 38.3 38.4 38.5 38.6


(155528)

0

20

40

60

80

100

Inte

nsity

13C-O8CDF / Average

Manual Method with ASE Automated Method with BUCHI

• Blank chromatograms

FFGFG FFGFG

5. Results


SpeedExtractor combined with Miura system

“scale from – to + in benefit”

6. Conclusions : advantages / drawbacks

Volume of Solvent +++ ( less than 150 mL vs more than 800 mL)

Volume of N2 +++ >1000 samples with a tank vs 100

Time for Extraction/ Purification

++ (3,5h vs 5,5h)

Numbers of samples per day ++

Repeatability +++

Cross Contamination ++

Price + or maybe +++ if you are a good negociator

Drawbacks ???

Other methods VS


“Extra slides…”

For 14 samples

For 14 samples


“Extra slides…”


http://www.laberca.orghttp://www.saraf-educ.org

Acknowledgement

Documents

lnnovative strategy for Dioxins/PCBs extraction and ...Vincent Vaccher| RAFA 2015| 04-11-2015| PRAGUE 4/32 1. LABERCA in few slides... •Research Department of Oniris (Nantes-Atlantic