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Saskia S. SterkHenk A. Herbold,
October 2006.
EVALUATION AND INNOVATION OF OUR PROFICIENCY TESTING PROGRAM.
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1.
CRL workshop 2005 2ARO: Food and ResiduesARO: Food and Residues
Content of the presentation.
1. Evaluation of the results, proficiency tests 2004 - 2006.
2. Overview of the current proficiency tests (2006).
3. Proficiency testing program: ‘future plans’.
4. Additional proficiency test information on the website.
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CRL workshop 2005 3ARO: Food and ResiduesARO: Food and Residues
(1) Overview proficiency tests period 2004 – 2006.
11015September 2006
(7) Ethynylestradiol in lyophilized bovine urine.
41725June 2006(6) Diethylstilbestrol in lyophilized bovine urine.
51117November 2005
(5) Methylboldenone in lyophilized bovine urine.
-610August 2005(4) Metabolism and analytical methodology for 17ß-norclostebol. *(Research-study)
31420August 2005(3) Trenbolone in lyophilized bovine urine.
24December 2004
(2a) Zeranol and Taleranol in samples of lyophilized bovine ‘bile’.
41527December 2004
(2) Zeranol and Taleranol in samples of lyophilized bovine ‘urine’.
3912June 2004(1) Norethandrolone and metabolites in lyophilized bovine urine.
No resultsNRLs*ParticipantsPeriodProficiency tests
* For EU maximum number of NRLs is 24 (NRL for Luxemburg is NRL for Belgium)
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CRL workshop 2005 4ARO: Food and ResiduesARO: Food and Residues
94023.0(low concentration)
93625.9(high concentration)
Norethandrolone
NCV%False negativeMean of all (ppb)
2498160.5(Taleranol)
2491150.6(Zeranol) (low concentration)
Zeranol and Taleranol (urine)
244517.2(Taleranol)
245713.0(Zeranol) (high concentration)
Zeranol and Taleranol (urine)
126530.4Batch B
129330.7Batch A
Methylboldenone
2541-1.0(Trans-Des / low concentration)
2533-3.7(Trans-Des / high concentration)
Diethylstilbestrol
(2) Overview proficiency tests period 2004 – 2006.
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CRL workshop 2005 5ARO: Food and ResiduesARO: Food and Residues
Possible reasons of the variation.
• Hydrolysis step (temperature and time)
• Standards (Purity)
• The use of an internal standard (Deuterated).
• Detection technique (GC-MS, LC-MS)
• Mass concentration
• Validated method (reproducibility, repeatability, CCα, CCß)
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CRL workshop 2005 6ARO: Food and ResiduesARO: Food and Residues
Overview confirmation.
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CRL workshop 2005 7ARO: Food and ResiduesARO: Food and Residues
Conclusion.
•Most of the participating Labs (include NRLs) had no problems in confirming the analyte.
•The results of the proficiency tests accentuated some problems regarding the quantification of the analyte.
•It is obvious that quantification of low concentrations gave more variations in the results.
•Information of the used in-house method in detail, is interesting. Because it is the only way to screen for factors which could have an effect on the results.
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CRL workshop 2005 8ARO: Food and ResiduesARO: Food and Residues
(2) Evaluation of the results, proficiency testing program 2006.
• Results RIVM/CRL proficiency test Diethylstilbestrol in lyophilized bovine urine. Report 310309 016 2006 (June 2006)
• Results RIVM/CRL proficiency test Ethynylestradiol in lyophilized bovine urine. Report 310309 017 2006 (September 2006)
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CRL workshop 2005 9ARO: Food and ResiduesARO: Food and Residues
Results RIVM/CRL proficiency test Diethylstilbestrol in lyophilized bovine urine. Report 310309 016 2005
9
* Total number of participants included the NRL
CRL workshop 2005 10ARO: Food and ResiduesARO: Food and Residues
Diagram excretion of trans- and cis-DES after treatment of animal and with different enzyme preparations.
Excretion trans-Des (free, SHP, Gluc)
-500
0
500
1000
1500
2000
2500
0 1 2 3 4 5 6 7 8 9 10 11 12 13
days
conc
entr
atio
n (p
pb)
free
shp
gluc
Excretion cis-DES (free/SHP/Gluc)
-200
0
200
400
600
800
1000
1200
0 1 2 3 4 5 6 7 8 9 10 11 12 13
days
conc
entr
atio
n (p
pb)
free
shp
gluc
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CRL workshop 2005 11ARO: Food and ResiduesARO: Food and Residues
Samples prepared for this study.
• Batch A. High concentration of approximately 2.5 ppb of trans-DES and cis-DES.
• Batch B. Low concentration of (approximately 1 ppb) of trans-DES and cis-DES.
• Batch C. High concentration of approximately 9 ppb of DES-glucuronide.
• Batch D. Blank samples of urine.
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CRL workshop 2005 12ARO: Food and ResiduesARO: Food and Residues
Results RIVM/CRL proficiency test Diethylstilbestrol in lyophilized bovine urine. Report 310309 016 2006
Trans Cis
DES - isomerization
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CRL workshop 2005 13ARO: Food and ResiduesARO: Food and Residues
DES (di-TMS derivative). (Diagnostic ions 368, 383, 397 and 412 m/z).
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1 ulInjection volume
GC-MS/EI (368, 383, 397, 412 m/z)Ionization
80 °C for 1 min, increased at 20 ° C/ min. to 300 °C and held at this temp. for 5 min.
Oven-temperature
30 meter VF-5MS, Low Bleeding/ non polar fused silica capillary (0.25 mm i.d. 0.2 μm film)
Column
Helium Carrier gas
CRL workshop 2005 14ARO: Food and ResiduesARO: Food and Residues
Overview of analytical results of all laboratories.
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CRL workshop 2005 15ARO: Food and ResiduesARO: Food and Residues
Graphical representation scores for NRLs. (1 indicates “no participation”)
15
Unacceptable Low Good High
CRL workshop 2005 16ARO: Food and ResiduesARO: Food and Residues
Comparing first and second measurement (Day 1)(29 participants - include NRLs).
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CRL workshop 2005 17ARO: Food and ResiduesARO: Food and Residues
Comparing the characteristics of the method.‘Hydrolysis’ (1).
2x17
1x7x1x16
1x15
2x1x3
1x1x1x2x5x2
62°C60°C55°C52°C50°C40°C37°C20°CHours
Interval of hydrolysis.
02468
1012
2 3 15 16 17
Hours
Num
ber
of p
artic
ipan
ts
17
CRL workshop 2005 18ARO: Food and ResiduesARO: Food and Residues
Comparing the characteristics of the method.‘Hydrolysis’ (2).
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CRL workshop 2005 19ARO: Food and ResiduesARO: Food and Residues
Comparing the characteristics of the method.‘Hydrolysis’ (3). (Spiked with Des-glucuronide)
Hydrolysis process.trans-DES (2005M2460 – Batch C)
0,00
1,00
2,00
3,00
4,00
5,00
3 hours40°C
2 hours37°C
2 hours62°C
Overnight52°C
Overnight20°C
2 hours60°C
Overnight37°C
3 hours55°C
Time and temperature (pH 4.8 - 5.5).
Yie
ld (p
pb)
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CRL workshop 2005 20ARO: Food and ResiduesARO: Food and Residues
Confirmation.
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CRL workshop 2005 21ARO: Food and ResiduesARO: Food and Residues
Conclusion.
• The participated NRLs was 67%. For an analyte, routine for many years, is this participation is too low.
• Most of the laboratories reported to be able to confirm residuesaccording to the EU criteria, (Commission Decision 2002/657/EC).
• Results. Four laboratories reported excellent results (score 18-20). One laboratory reported good results (score 15-17) and 5 laboratories reported acceptable results (score 12-14).
• Most of the participants used for detection GCMS or GCMS/MS. Only two laboratories sent us results obtained by LCMS/MS.
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CRL workshop 2005 22ARO: Food and ResiduesARO: Food and Residues
Results RIVM/CRL proficiency test Ethynylestradiollyophilized bovine urine. Report 310309 017 2006 (September 2006)
Number of participants that didn’t send us any results.11No results
Number of participants that submitted confirmatory results.
1015Confirmed
Number of participants that submitted any results.1015Analysed
NRLParticipants*
* Total number of participants included the NRL.
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CRL workshop 2005 23ARO: Food and ResiduesARO: Food and Residues
Samples were diluted with blank bovine urine to the target concentrations.
• Batch A. A mix of urine samples with a “high” concentration of approximately 7.5 ppb of Ethynylestradiol.
• Batch B. A mix of urine samples with a “low” concentration of approximately 5 ppb of Ethynylestradiol.
• Batch C. Blank samples of urine.
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CRL workshop 2005 24ARO: Food and ResiduesARO: Food and Residues
Ethynylestradiol (di-TMS derivative). Diagnostic ion 440,425,300,285 m/z.
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CRL workshop 2005 25ARO: Food and ResiduesARO: Food and Residues
Evaluation performance individual scores laboratories.
7212243
1010040
72122*3872122*3372122*3210010*31
7212230
5012228
6212126
52111*22
7212220
72122*1872122*1752111*1572122*10
TotalConf.2005M10982005M10972005M1096NRL
67 % of the participants make a score of 6 or 7.
20 % of the participants make a score of 5.
13 % of the participants make a score of 1.
25
Score NRLs
012345678
10 15 17 18 22 31 32 33 38
Lab codes
ToT
al sc
ores
CRL workshop 2005 26ARO: Food and ResiduesARO: Food and Residues
Comparing the characteristics of the method.‘Hydrolysis’ (1).
118
117
3116
115
13
11222
62°C60°C55°C52°C40°C37°C20°CHours
Interval of hydrolysis.
01234567
2 3 15 16 17 18
Hours
Num
ber
of p
artic
ipan
ts
26
CRL workshop 2005 27ARO: Food and ResiduesARO: Food and Residues
Comparing the characteristics of the method.‘Hydrolysis’ (2).
Hydrolysis process.EE2 (2005M1096 – Batch A).
0,002,004,006,008,00
10,0012,0014,00
2 hours 37°C 3 hours 40°C 2 hours 62°C overnight 20°C Overnight 37°CTime and temperature (pH 4.8 - 5.5).
Yie
ld (p
pb)
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CRL workshop 2005 28ARO: Food and ResiduesARO: Food and Residues
Confirmation.
-++43
---40
-++38
-++33
-++32
---31
-++30
n.a.n.a.n.a.28
-++26
-++22
-++20
-++18
-++17
-++15
-++10
2005M10982005M10972005M1096Labcodes
28
CRL workshop 2005 29ARO: Food and ResiduesARO: Food and Residues
Conclusion.
• The average values obtained by the participants were very close to the target concentrations. The majority of participants reported values within 1 SD (approx. 35%) of this overall average.
• Most of the laboratories reported to be able to confirm residues of EE2 according to the EU criteria, (Commission Decision 2002/657/EC).
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CRL workshop 2005 30ARO: Food and ResiduesARO: Food and Residues
Proficiency testing program 2007 – 2008.
Metabolites of 19Nor-4-androsteen 3,17 dion in samples of bovine urine. (Ongoing - report probably February 2007)
Oestradiol in samples of bovine urine and plasma. (Ongoing - report probably January 2007)
Gestagens in samples of porcine kidney fat. (Spring 2007)
Thyreostatica in samples of porcine urine. (Autumn 2007)
Proficiency testing program for 2008 (to be under discussion)
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CRL workshop 2005 31ARO: Food and ResiduesARO: Food and Residues
The way forward.
http://www.rivm.nl/residues
Our new service is to provide additional information, about reference
materials and proficiency tests on the website.
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CRL workshop 2005 32ARO: Food and ResiduesARO: Food and Residues
Thank you for your attention.
Thank you for your attention.
32