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

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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

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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

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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

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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

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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

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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

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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

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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

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Example spectrum NCI a-zearalanol, standard 0.1 ng absolute

11FAIR RAL’s project (Okt 2002) | Marco Blokland

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Example spiked urine on 0.1 ng/ml

12FAIR RAL’s project (Okt 2002) | Marco Blokland

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Example continued

13FAIR RAL’s project (Okt 2002) | Marco Blokland

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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