FAIR 5 PL97-3443 project Stability study GCMS analysis ... · GC-MS analysis • Goal development...
of 22
/22
Embed Size (px)
Transcript of FAIR 5 PL97-3443 project Stability study GCMS analysis ... · GC-MS analysis • Goal development...
RE 1
Stability studyGCMS analysisGCMS ringtest
FAIR 5 PL97-3443 project
O
HO
OH CH3
O
O
Zearalanone
O
HO
O H CH3O
O H
O
HO
OH CH3O
OH
O
HO
O H CH3O
OH
O
HO
OH CH3O
OH
O
HO
OH CH3
O
O
ZeranolTale ranol
Zearale none
ß-Zearalenol α -Zearalenol
? ?
Fusariumtoxins
Zeranol &metabolites
G. Kennedy et al. Food Additives and Contaminants. 1998. vol 15. No 4. 393-400
2FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Stability Study
• Goal, preparation of reference material
• Blank urine’s used are from non treated animals
• Alpha- and beta-zearalenol and Zeranol in urine, bile from cattle and urine from sheep and pig
• On “natural” levels 3 ng/ml α-zearalanol, 30 ng/ml α/ß-zearalenol
• On “abuse” levels 30 ng/ml α-zearalanol, 30 ng/ml α/ß-zearalenol
3FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Design stability study
Bile
Urine
Cattle
Cattle
Sheep
PigNatural
Abuse-20°C frozen
-20°C lyop.
+4°C lyop.
T=0
T=1 week
T=2 weeks
T=4 weeks
T=8 weeks
T=16 weeksSamples were analysed in duplicate
4FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Statistical model
Cattle urine -20°C frozen, zeranol
60
80
100
120
140
0 3,0 7,0 11,0 15,0Time (weeks)
Nor
mal
ised
to -2
0°C
ly
ophi
lised
pig
urin
e (%
)
1,08
1,96
0,21
94,0
-20%
+20%
5FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Results stability study
ID Condition α-zearalanol α-zearalenol ß-zearalenolCattle Urine -20°C Frozen 22 - * 11 - * 19 - *
-20°C Lyophilised 18 - * 12 - * 11 – 52+4°C Lyophilised 12 – 176 6 – 20 7 – 18
Cattle Bile -20°C Frozen * - * 6 - * 14 - *-20°C Lyophilised * - * 11 - * 10 - *+4°C Lyophilised * - * 7 - * 10 - *
Sheep Urine -20°C Frozen 13 - * 11 - * 19 - *-20°C Lyophilised 14 - * 7 - * 18 - *+4°C Lyophilised 7 – 41 7 – 96 6 –13
Pig Urine -20°C Frozen 19 - * 8 - * 7 - *-20°C Lyophilised 11 – 140 6 – 174 8 – 13+4°C Lyophilised 13 – 74 6.3 - * 13 - *
* stability time infinitive (stable)
6FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Summarised results stability studyID Condition α-zearalanol α-zearalenol ß-zearalenol
Cattle urine -20°C frozen Stable Stable Stable
-20°C lyophilised Stable Stable Not stable
+4°C lyophilised Not stable Not stable Not stable
Cattle Bile -20°C frozen Stable Stable Stable
-20°C lyophilised Stable Stable Stable
+4°C lyophilised Stable Stable Stable
Sheep urine -20°C frozen Stable Stable Stable
-20°C lyophilised Stable Stable Stable
+4°C lyophilised Not stable Not stable Not stable
Pig urine -20°C frozen Stable Stable Stable
-20°C lyophilised Not stable Not stable Not stable
+4°C lyophilised Not stable Stable Stable
7FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Conclusions stability study
– Cattle urine and pig urine at +4°C lyophilised will cause a
reduction of the content of all analyts, there is also a
reduction in the α-zearalanol content in sheep urine, other
compounds are stable
– At -20°C lyophilised reduction of ß-zearalanol in cattle and
pig urine, and also of α-zearalenol in pig urine.
– Material is stable at frozen condition.
– Lyophilised materials are available for validation .
8FAIR RAL’s project (Okt 2002) | Marco Blokland
r
GC-MS analysis
• Goal development of method able to detect all RAL’s
• One method for all matrices, urine, liver, meat, kidney and bile
• Validation of the analytical method following SANCO/1085/2000 guidelines
9FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Flow chart of analytical method
URINE - BILE - LIVER - KIDNEY MEAT
HydrolysisSuc. Helix
Enzymaticdigestion
Liquid - Liquid Extraction
SPEC18 and NH2
GC-MS (NCI)
10FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Example spectrum NCI a-zearalanol, standard 0.1 ng absolute
11FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Example spiked urine on 0.1 ng/ml
12FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Example continued
13FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Determination (n=1)of the CCα and CCß (ng/ml), urine
Analyts CCα CCßZeranol 0.06 0.11Taleranol 0.07 0.12α-zearalanol 0.07 0.11ß-zearalanol 0.21 0.36 zearalenone 0.35 0.60 zearalanone 0.19 0.33
ID α-lanol ß-lanol α-lenol ß-lenol lenone lanoneAverage 71.7 67.8 65.5 71.5 37.5 86.9CV (%) 1.8 3.3 0.4 3.6 5.5 11.9
Determination (n=3)of the recovery (%), urine
14FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Repeatability, accuracy (%) ( 5 days, three analyses) in urine samples spiked at 1 μg/l
ID Zeranol Taleranol α-zearalenol ß-zearalenol zearalenone zearalanoneAverage 95.3 103.1 153.6 88.3 65.7 87.7CV (%) 2.1 4.1 2.9 3.0 4.2 2.9Average 107.3 106.6 113.6 113.6 99.5 80.1CV (%) 2.7 1.4 2.2 3.9 9.4 9.8Average 114.5 111.3 120.9 130..9 69.5 109.1CV (%) 3.3 0.9 3.8 2.8 5.5 7.9Average 101.2 100.5 134.2 119.1 89.2 114.2CV (%) 4.0 4.1 6.2 2.4 8.6 8.2Average 118.2 118.1 153.8 130.2 69.9 155.4CV (%) 5.3 3.0 5.0 2.0 5.7 2.2Average 107.3 107.9 135.2 116.4 78.8 107.5CV (%) 8.7 6.5 13.1 14.0 18.5 27.5
15FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Comparison of the results obtained by spiking urine (n=10) and liver (n=10) at 0.5 μg/l
ID Zeranol Taleranol α-zearalenol ß-zearalenol zearalenone zearalanoneUrine Liver Urine Liver Urine Liver Urine Liver Urine Liver Urine Liver
Average 90.4 86.6 94.4 91.1 160.8 164.9 100.9 134.5 75.7 91.5 63.5 95.7CV (%) 2.9 3.1 2.2 2.9 20.2 16.9 2.8 3.9 63.9 64.6 66.2 57.5
Change to another matrix is considered as one major change, only supplementary validation is necessary
ID α-zearalanol ß-zearalanol α-zearalenol ß-zearalenol Zearalenone ZearalanoneUrine Bile Urine Bile Urine Bile Urine Bile Urine Bile Urine Bile
Average 116.6 111.5 127.7 120.0 118.5 187.0 111.7 189.4 373.9 120.7 355.8 131.7CV (%) 6.2 5.4 5.0 6.2 13.3 3.8 4.4 4.5 36.5 38.5 36.4 45.3
Comparison of the results obtained by spiking urine (n=10) and bile (n=10) at 0.5 μg/l
16FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Correlation of GC-MS (CRL) vs. LC-MSMS (VSD)
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120 140
GC-MS (CRL) (ng/ml)
LC-M
SMS
(VSD
) (ng
/ml)
a-zearalanolß-zearalanola-Zearalenol ß-zearalenol
17FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Correlation of GC-MS (CRL) vs. LC-MSMS (VSD)
0
2
4
6
8
10
12
14
16
18
20
0 5 10 15 20
GC-MS (CRL) (ng/ml)
LC-M
SMS
(VSD
) (ng
/ml)
a-zearalanolß-zearalanola-Zearalenol ß-zearalenol
18FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Conclusions validation analytical method
– Difference in performance [α/ß-zearalanol, α/ß-zearalenol] and
[zearalenone, zearalanone].
– No differences in performance between matrices for α/ß-
zearalanol.
– For the other compounds the difference is significant.
– Quantitative method for α/ß-zearalanol and α/ß-zearalenol.
– Qualitative method for zearalanone, zearalenone
– Confirmation by use of GC-MS (EI) or LC-MSMS
19FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Ringtest
• Goal, harmonisation of GC-MS methods used
Prepared samples (lyophilised)
ID α-zearalanol ß-zearalanol α-zearalenol ß-zearalenol Zearalenone Zearalanone
A Blank Blank blank blank Blank blankB Blank Blank 10 ng/ml 10 ng/ml Blank blankC 2 ng/ml 2 ng/ml blank blank blank blankD 2 ng/ml 2 ng/ml 10 ng/ml 10 ng/ml 2 ng/ml 10 ng/mlE 5 ng/ml 5 ng/ml 5 ng/ml 5 ng/ml 5 ng/ml 5 ng/ml
20FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Overview results ringtest
0
2
4
6
8
10
12
14
16
A B C D E A B C D E A B C D E A B C D E A B C D E A B C D E
Con
cent
ratio
n (n
g/m
l)
RIVMPortugalSwedenIreland
α-zearalanol zearalanonezearalenoneß-zearalanolα-zearalenolß-zearalanol
21FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Conclusions ringtest
– Overall variability a/ß-zearalanol and a/ß-zearalenol was 10-35% for
all tested concentrations
– Variability zearalenone and zearalanone higher
22FAIR RAL’s project (Okt 2002) | Marco Blokland
r
Last remarks
• Incurred RAL’s materials (urine and bile) are available to
the NRL’s
• Developed method, validation results, ringtest, comparison
results VSD/CRL will published near future
