Annual CRL-ISS Workshop 2009
on Proficiency Tests
“PREPARATION OF PT MATERIALS”
Maria Ciprotti
CRL-ISS
CRL-ISS Annual Workshop 23 october 2009
13th PT 1° round: Milk
Milk Plan
Purchasing of material
Spiking +
homogenization
Level A
µg/l
Level B
µg/l
Pb
~ 35
~ 22
Cd
~ 7.5
~ 13
As
~ 75
~ 40
CRL-ISS Annual Workshop 23 october 2009
13th PT 1°round: Milk
A liquid milk sample was prepared by using a product
from the market, in order to allow the laboratories to
apply their methods in routine conditions.
Partition into two aliquots
Spiking of As, Cd, Pb at two concentration levels
Homogenization by a peristaltic pump
CRL-ISS Annual Workshop 23 october 2009
13th PT 1°round:Milk
Sampling by a peristaltic pump
Sterilization process in autoclave (3 min, 120°C + quenching)
.:
Associazione Italiana Allevatori ( Italian Breeder Association)
Labelling
Evaluation of the
Material homogeneity
(IUPAC 2006)
Shipping
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
Shipment of freeze-dried material with a content of:
As ~ 1.75 mg/Kg
Cd ~ 0.30 mg/Kg
The step related to lyophilization
Pb ~ 0.30 mg/Kg
concentrates the content of the elements
Hg~ 1.23 mg/Kg
-
increase of elements of interest during lyophilization
endogenous content of As and Hg
to detect the species of fish able to mix together in a homogeneous way
These considerations forced us to perform preliminary tests as follows.
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
Preliminary Test
Analyses of different species
of fish (sea and fresh water)
As
(µg/kg)
Cd
(µg/kg)
Pb
(µg/kg)
Hg
(µg/kg)
Perch
< 42.5
<2
<3
49
Swordfish
1315
6.0
<3
392
Salmon
225
<2
<3
20
Pangasius
< 42.5
<2
<3
<2
From concentrations obtained and from the consistency of the materials, it was
decided to mix swordfish and pangasius. *
Swordfish
Pangasius fish
Italian word
Italian word
Pescespada
Pangasio
SPADASIO
CRL-ISS Annual Workshop 23 october 2009
Swordfish just
purchased
(frozen)
13th PT 2°round: Fish
Preliminary Test
Frozen water
removed
Distributed to be
frozen again
later
Non edible parts
removed
Blended
Roughly minced
Pangasius undergoes the
same identical process
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
Preliminary Test
After homogenization of each species, the material was mixed in proportions
so as to obtain the expected value of the elements after lyophilization.
During mixing the spiking of the elements Cd and Pb was performed using
standard solutions prepared gravimetrically.
After spiking and homogenization, the test material
was sent to a pilot lyophilizer calibrated on the
characteristics of the lyophilizing system to be used
later for the final bulk.
Portions of the fish species without spiking of the
elements are lyophilized to verify the yield of the
process of lyophilization of each species.
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
Preliminary Test
Lyophilized samples are blended and mixed
and afterwards sieved (size < 500 µm)
From the three samples obtained
• spadasio
• swordfish
• pangasius
aliquots are taken
concentrations
to
verify
the
obtained
The results of these analyses confirm the calculation carried out to obtain the
concentrations of interest of the elements As, Cd, Pb e Hg
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
Preliminary Test
Swordfish
Pangasius fish
Spadasio
Asmes : 0.894 ppm
Cdmes :0.0396 ppm
Pbmes : <0.003 ppm
Hgmes :0.680 ppm
Asmes : <0.042 ppm
Cdmes : 0.005 ppm
Pbmes : 0.005 ppm
Hgmes : <0.002ppm
Lyoph. yield
25%
Lyoph. yield
15.6%
Asmes : 3.657 ppm
Cdmes : 0.166 ppm
Pbmes : 0.028 ppm
Hgmes :2.683 ppm
Asmes : 0.188 ppm
Cdmes : 0.005 ppm
Pbmes : 0.034 ppm
Hgmes : <0.002 ppm
Lyoph. yield
19.6%
Asmes : 2.013 ppm
Cdmes : 0.196 ppm
Pbmes : 0.39 ppm
Hgmes : 1.481 ppm
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
Preliminary Test
Fresh Swordfish
Fresh Pangasius
Hg mes.
Asmes
Cdmes.
Pbmes.
Hgmes.
Asmes.
Cdmes.
Pbmes.
0.68
0.894
0.04
<LoQ
<LoQ
<LoQ
0.005
0.003
Lyophilized Swordfish
Hg mes. Hg calc As mes. As calc. Cd mes. Cd calc. Pb mes. Pb calc.
2.683
2.712
3.657
3.566
0.166
0.158
0.028
<LoQ
Lyophilized Pangasius
As mes.
As calc.
Pb mes.
Pb calc.
0.188
0.1536
0.034
0.022
Lyophilized spadasio
Hg mes.
Hg calc
As mes.
As calc.
Cd mes.
Cd calc.
Pb mes.
Pb calc.
1.481
1.485
2.013
2.080
0.196
0.199
0.39
0.408
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
After obtaining the test sample with the expected concentrations, we proceed
with the preparation of the bulk material in the same way used in the previous
procedure; this step can be synthesized as follows:
1)
2)
Cleaning of
the fish
3)
Homogenization
4) Distribution in
Mixing of different
species + spiking
containers
to be sent
for lyophilization
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
Mixing
Sieving
Quartering and
bottling
γ-Irradiation
CRL-ISS Annual Workshop 23 october 2009
Preparation
Swordf. Pang
485
1000
g fresh sample
Concentration
factor of lyophilized 0.2503
sample
0.156
g lyophilized sample 121.4
156
Cd
Pb
**
~ 60
~ 75
H2O
300
13th PT 2°round: Fish
**µg to be spiked at each
1485g of spadasio; they
will be present in the 100g
of H2O
g of the 100ppm to be
added to 100g H2O
~ 0.6
~ 0.75
Concentration
mg/kg
mg/kg mg/kg hypothesis hypothesis mg/kg:
Swordf. Pangasius Spadasio Spadasio + moisture
Consensus values
(mg/kg)
As
3.657
0.166
1.69
1.74
As
n.e.
Cd
0.166
< LoQ
0.289
0.30
Cd
0.281
Hg
2.71
0.01
1.192
1.23
Hg
1.25
Pb
< LoQ
0.034
0.291
0.30
Pb
0.276
CRL-ISS Annual Workshop 23 october 2009
13th PT 2°round: Fish
When mixing two different species of fish, homogenization could not be
sufficient and therefore the material could be not fit for purpose.
As previously indicated, the homogeneity test is carried out in accordance with
the “Harmonised Protocol” (IUPAC 2006).
The homogeneity test for As, Cd, Pb and Hg is performed on about 0.5 g of the
material:
H: σ2sam < σ2all
CRL-ISS Annual Workshop 23 october 2009
Additional analyses
PT / year
13th / 09
12th / 08
12th / 08
11th / 07
11th / 07
Sample
Fish muscle
Bovine meat
Bovine liver
Fish muscle
Pork meat
Results
Pb
Conc. SD%
0.286
7.3
0.165
9.1
0.553
4.0
0.225
4.9
0.113
8.8
13th PT 2°round: Fish
Cd
Conc. SD%
0.315
6.2
0.07
7.1
0.589
0.8
0.018
5.5
0.034
7.1
High-Resolution continuum source graphite
furnace AAS ContrAA+ automatic solid
autosampler SSA600 (Analytikjena)
Hg
Conc. SD%
1.220
1.64
0.753
1.29
Direct mercury
analyzer DMA 80
(CRL-ISS)
CRL-ISS Annual Workshop 23 october 2009