Click to edit Master title style · Saskia S. Sterk Henk A. Herbold, October 2006. EVALUATION AND...

Saskia S. SterkHenk A. Herbold,

October 2006.

EVALUATION AND INNOVATION OF OUR PROFICIENCY TESTING PROGRAM.

Click to edit Master title style

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.

2


(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)

3


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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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)

5


Overview confirmation.

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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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(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)

8


Results RIVM/CRL proficiency test Diethylstilbestrol in lyophilized bovine urine. Report 310309 016 2005

9

* Total number of participants included the NRL


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

10


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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Results RIVM/CRL proficiency test Diethylstilbestrol in lyophilized bovine urine. Report 310309 016 2006

Trans Cis

DES - isomerization

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DES (di-TMS derivative). (Diagnostic ions 368, 383, 397 and 412 m/z).

13

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


Overview of analytical results of all laboratories.

14


Graphical representation scores for NRLs. (1 indicates “no participation”)

15

Unacceptable Low Good High


Comparing first and second measurement (Day 1)(29 participants - include NRLs).

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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



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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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Confirmation.

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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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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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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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Ethynylestradiol (di-TMS derivative). Diagnostic ion 440,425,300,285 m/z.

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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



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



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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Confirmation.

-++43

---40

-++38

-++33

-++32

---31

-++30

n.a.n.a.n.a.28

-++26

-++22

-++20

-++18

-++17

-++15

-++10

2005M10982005M10972005M1096Labcodes

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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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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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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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Thank you for your attention.

Thank you for your attention.

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Click to edit Master title style · Saskia S. Sterk Henk A. Herbold, October 2006. EVALUATION AND...

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