PLE for Extraction of Dioxins in
Animal Feed and Ingredients
J F Focant G Scholl E De PauwJ.-F. Focant, G. Scholl, E. De Pauw,and G. Eppe
MS LaboratoryMS LaboratoryBiological and Organic Anal. Chem.
Belgian Chicken Gate 1999
Animal feedingstuffsAnimal feedingstuffs
Concentration Factor
EU Guidelines
Key Points of EU Guidelines ISO/CEI 17025
LOQs < 20% target level
Bl k QC CRM t i ifi i t l Blanks, QCs, CRMs, matrix-specific intercal.
CVs < 15%, justness ±20%, recovery rates %…%, j %, y %
GC-IDHRMS
Upperbound ≠ lowerbound < 20-40%
C li t EPA 1613b th i l t Compliant EPA 1613b or other equivalent…
Organohalogen Compounds, 2008, 70, 902.Issues
Need for Harmonization… Should it be a single method ?
Sh ld l b b ‘f ’ t i h th d ? Should labs be ‘free’ to use in-house methods ?
Manual vs automated ?
What in case of conflict ?
Methods inter-compatibility , ‘transportability ‘?
St d d th d ( f b d) ? Standard method (performance-based) ?
CEN method to come…
Intercalibration exercises (matrix-specific) ?
Automated ApproachAutomated extraction
Automated clean-up
A t t d f ti tiAutomated fractionation
Automated GC-MS injectionj
Sequential PLE (ASE)Pump
O
Extraction
Solvents Oven
Cell
CollectionCollectionVial
Extraction Cell
S iki Spiking Homogenization D i (l h ) Drying (lyoph, …) 120°C, 1500 psi H t l EtOH
N SO
Hexane, toluene, EtOH, … Static and/or dynamic
Na2SO4
10-30 min/sample
Parallel PLE
E t tiExtraction,solvent
reduction andand
exchange
Parallel Clean-up and Fractionation
Focus on ExtractionASE (Dionex) : Sequential extractions
PLE (FMS) : Parallel (6) extractions
CEN recommended conditions (PT):
100°C, 1500 psi, 15 min static
Flush with solvent, purge with nitrogen
3 cycles (C1 = Tol C2-3 = Tol/EtOH 90:10)3 cycles (C1 Tol, C2 3 Tol/EtOH 90:10)
Mineral Clay PCDD/F Profile
1 21,41,61,82,0
oduc
t ASEPLE
0,20,40,60,81,01,2
ng/k
g pr
o
CEN Concensus
0,0,
Mineral Clay DL-PCB Profile
40
25
30
35
40
uct
ASEPLECEN Concensus
10
15
20
25
ng/k
g pr
od CEN Concensus
0
5
ortho)
ortho
)
ortho
)
ortho
)
rtho)
rtho)
rtho)
rtho)
rtho)
rtho)
rtho)
rtho)
PCB 77 (n
on-or
tPCB 81
(non
-ort
PCB 126 (
non-o
rtPCB 16
9 (no
n-ort
PCB 105 (
ortPCB 11
4 (ort
PCB 118 (
ortPCB 12
3 (ort
PCB 156 (
ortPCB 15
7 (ort
PCB 167 (
ortPCB 18
9 (ort
Mineral Clay TEQ
0,8
0 5
0,6
0,7
duct
ASEPLE
0,3
0,4
0,5
g TE
Q/k
g pr
od CEN Concensus
0,1
0,2
ng
0,0PCDDFs TEQ DL PCBs TEQ PCDDF+ DL PCBs TEQ
Bovine Feed TEQ
3,0
2,5
3,0
ct
ASEPLE
1,5
2,0
EQ/k
g pr
oduc CEN Concensus
0,5
1,0
ng T
E
0,0PCDDFs TEQ DL PCBs TEQ PCDDF+ DL PCBs TEQ
Fish Meal TEQ
1,6
1,8
2,0
ASEPLE
1,0
1,2
1,4
Q/k
g pr
oduc
t
CEN Concensus
0,4
0,6
0,8
ng T
EQ
0,0
0,2
PCDDFs TEQ DL PCBs TEQ PCDDF+ DL PCBs TEQ
Z-Scoring PT (TEQ)Z-scores ConcensusASE PLE Level (ng/kg)
Mineral clay PCDD/Fs 1 4 0 9 0 56
Z-scores ConcensusASE PLE Level (ng/kg)
Mineral clay PCDD/Fs 1 4 0 9 0 56Mineral clay PCDD/Fs 1,4 -0,9 0,56PCBs 0,0 -2,5 0,04PCDD/Fs+PCBs 1,3 -1,0 0,60
Bovine feed PCDD/Fs 1 8 0 3 1 49
Mineral clay PCDD/Fs 1,4 -0,9 0,56PCBs 0,0 -2,5 0,04PCDD/Fs+PCBs 1,3 -1,0 0,60
Bovine feed PCDD/Fs 1 8 0 3 1 49Bovine feed PCDD/Fs 1,8 0,3 1,49PCBs 0,1 -0,3 1,02PCDD/Fs+PCBs 1,1 0,0 2,51
Fish meal PCDD/Fs 1 4 -2 6 0 58
Bovine feed PCDD/Fs 1,8 0,3 1,49PCBs 0,1 -0,3 1,02PCDD/Fs+PCBs 1,1 0,0 2,51
Fish meal PCDD/Fs 1 4 -2 6 0 58Fish meal PCDD/Fs 1,4 2,6 0,58PCBs 0,7 -0,7 0,84PCDD/Fs+PCBs 1,0 -1,5 1,42
Fish meal PCDD/Fs 1,4 2,6 0,58PCBs 0,7 -0,7 0,84PCDD/Fs+PCBs 1,0 -1,5 1,42
ASE positive bias ?Concensus value based on mainly Soxhlet…
PLE negative bias ?
Extraction Condition StudyCEN recommended conditions (PT):
100°C, 1500 psi, 15 min staticFlush with solvent, purge with nitrogenp g g3 cycles (C1 = Tol, C2-3 = Tol/EtOH 90:10)
CART conditions CART conditions
150°C, 1500 psi, 15 min static
Flush with solvent, purge with nitrogen
3 cycles (C1-2-3 = Tol/EtOH 90:10)
Animal Feed QC Belgium In-house animal feed QC for testing F B l i 1999 i i From Belgian 1999 crisis ‘Naturally’ contaminatedAt level close to the EU regulatory value To be certified later??? To be certified later??? Used to test the future CEN method Comparison between CEN and CART methods Comparison between ASE and PLE Comparison between ASE and PLE Preliminary data
1,2
Is 100°C enough ?
1
mean CEN-ASE (100°C)mean CART-ASE (150°C)mean CEN-PLE (100°C)
CART PLE (150°C)0,8
dw
mean CART-PLE (150°C)
0,4
0,6
pg/g
d
0,2
,
0
etraC
DDnta
CDDex
aCDD
exaC
DDex
aCDD
ptaCDD
(OCDD)
etraC
DFnta
CDFnta
CDFex
aCDF
exaC
DFex
aCDF
exaC
DFep
taCDF
eptaC
DF(O
CDF)/Fs T
EQ
2, 3,
7, 8 -
Tetr
1, 2,
3, 7,
8 - P
ent
1, 2,
3, 4,
7, 8 -
Hex
1, 2,
3, 6,
7, 8 -
Hex
1, 2,
3, 7,
8, 9 -
Hex
1, 2,
3, 4,
6, 7,
8 - H
ept
OctaCDD (O
2, 3,
7, 8 -
Tet
1, 2,
3, 7,
8 - P
en
2, 3,
4, 7,
8 - P
en
1, 2,
3, 4,
7, 8 -
Hex
1, 2,
3, 6,
7, 8 -
Hex
1, 2,
3, 7,
8, 9 -
Hex
2, 3,
4, 6,
7, 8 -
Hex
1, 2,
3, 4,
6, 7,
8 - H
ep
1, 2,
3, 4,
7, 8,
9 - H
epOcta
CDF (O
Somme P
CDD/F
Temperature Control StudyIs there T control-transfer differences?
No ASE T data because no measure possible No ASE T data because no measure possible…
Closed vessel type
PLE T data possible because ‘open’ design
PLE: T probe in EtGly/water mix insidePLE: T probe in EtGly/water mix inside
the extraction cell
Temperature Study on PLEPLE temperaure monitoring (100°C)
120
80
100
(°C
) ±±10%10%
60
80
erat
ure
PLE soft are reading
20
40
Tem
pe PLE software readingCell temp
02 4 6 8 10 12 14 16 18 20
Time (min)Time (min)
Temperature Study on PLEPLE temperaure monitoring (150°C)
180
120
140
160
(°C
)
80
100
120
erat
ure
PLE software reading
20
40
60
Tem
pe PLE software readingCell temp
0
20
2 4 6 8 10 12 14 16 18 20
Time (min)Time (min)
Heat Transfer Aspect
Efficient Heat TransferPLE temperaure monitoring (150°C)
180
PLE temperaure monitoring (150°C)
180
PLE temperaure monitoring (150°C)
180
120
140
160
(°C
)
120
140
160
(°C
)
120
140
160
(°C
) ±±10%10%
80
100
120
pera
ture
PLE software reading80
100
120
pera
ture
PLE software reading80
100
120
pera
ture
PLE software reading
Cell temp
20
40
60
Tem
p
Cell temp20
40
60
Tem
p
Cell temp
Cell temp corrected20
40
60
Tem
p Cell temp
Cell temp corrected
Cell temp corrected &
0
20
2 4 6 8 10 12 14 16 18 20
Time (min)
0
20
2 4 6 8 10 12 14 16 18 20
Time (min)
Cell temp corrected0
20
2 4 6 8 10 12 14 16 18 20
Time (min)
pcontact
’ Time (min)Time (min)Time (min)5’ pre-heat15’ extraction cycle
Take Home Message Extraction of feed is complex…
Automated extraction tools ok for animal feed
Careful study of the extraction temperature is Careful study of the extraction temperature is
needed
Is 100°C enough ?
Is what the software says really what the T is ?
A pre-heat step is neededA pre-heat step is needed
