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Click to edit Master title style EVALUATION AND INNOVATION OF OUR PROFICIENCY TESTING PROGRAM. Henk Herbold Leen van Ginkel Saskia Sterk October 2008

Content of the presentation. 1. Evaluation of the results, proficiency test ‘Gestagens in porcine kidney fat’ 2. Short evaluation of the results, proficiency test ‘Thyreostatics in samples of porcine urine’ 3. Proficiency testing program 2008 - 2009: ‘future plans’ 2 ARO: Food and Residues ARO: Food and Residues

Overview of the proficiency test “Gestagens in porcine kidney fat” October 2008 3 ARO: Food and Residues ARO: Food and Residues

‘’Molecular structure of natural progesterone and MPA ”. General information. 4 ARO: Food and Residues ARO: Food and Residues

Animal experiment, preparation of incurred sample material. Porcine Kidney fat 5 ARO: Food and Residues ARO: Food and Residues

Comparing of the analytical possibilities. Kidney fat Liquid/Liquid (ppb) LC-MSMS GC-MS SPE Defatting average 2.69 4.00 HPLC LCMS STD 0.8 0.9 Fractionation CV% 34 21 Alkaline Hydrolysis N 17 6 Derivatisation GCMS 6 ARO: Food and Residues ARO: Food and Residues

Samples of porcine kidney fat, prepared for this study. • Batch A. High concentration (approximately 5 ppb) of MPA. • Batch B. Low concentration (approximately 1 ppb) of MPA. • Batch C. Blank samples. 7 ARO: Food and Residues ARO: Food and Residues

Homogeneity study. Single Mix Single Mix Batch A Batch A Batch B Batch B 1 5.6 4.5 1.8 1.1 2 6.9 4.4 1.4 1.1 3 5.1 4.8 1.4 1.1 4 6.2 4.5 1.4 1.1 5 5.5 4.2 1.6 1.0 6 6.6 4.7 1.4 0.8 Average 6.0 4.5 1.5 1.0 STD 0.7 0.2 0.2 0.1 CV% 11.6 4.7 11.2 11.7 F-test 6.060 0.906 Critical value of f (p=0.05) is 6.256 (N-1) 8 ARO: Food and Residues ARO: Food and Residues

Stability study Batch A 10 9 8 Concentrations (ppb) 7 4 °C 6 5 20 °C 4 37 °C 3 2 1 0 0 1 2 4 6 8 Weeks Batch B 1,8 1,6 Concentrations (ppb) 1,4 1,2 4 °C 1 20 °C 0,8 37 °C 0,6 0,4 0,2 0 0 1 2 4 6 8 Weeks 9 ARO: Food and Residues ARO: Food and Residues

Preparation of test materials, conclusions • The use of the LCMS method is preferred, because in case of GCMS, an alkaline hydrolysis and derivatisation step is necessary. • Comparison of the CV values of the samples single and mixed by F-test, shows no significant differences. Therefore the homogeneity is acceptable. • The stability is acceptable (CV<20%), with no significant differences between storing conditions, during the period of testing. 10 ARO: Food and Residues ARO: Food and Residues

Total number of participants Participants NRL Remarks (Total 30) Analysed 28 21 Number of participants that submitted results. Confirmed 25 Number of participants that submitted confirmatory results. No results 2 Number of participants that didn’t send us any results. 11 ARO: Food and Residues ARO: Food and Residues

Analytical steps in the procedures used by the participants. Quantification Labs • Liquid/Liquid extraction all • Solid Phase Extraction all • LC-MS or LCMS-MS 16 • GC-MS, GC-HRMS 8 • Elisa-kit 4 12 ARO: Food and Residues ARO: Food and Residues

Analytical steps in the procedures used by the participants. Confirmation Labs • Liquid/Liquid extraction all • Solid Phase Extraction all • LC-MS or LCMS-MS 18 • GC-MS, GC-HRMS 7 13 ARO: Food and Residues ARO: Food and Residues

Results MPA in Batch A. ppb Std CV% ppb Sample Batch A 7,00 CRL concentration: 4.57 Av1 6,00 Av2 5,00 Mean all laboratories (n=27) 3.59 1.13 32 Median 3.88 CRL value 4,00 conc. Trimmean 3,00 2*horwitz Mean all laboratories day 1 (n=27) 3.57 1.15 32 Max. 6.00 2,00 2*horwitz 1,00 Mean all laboratories day 2 (n=24) 0,00 3.70 1.11 30 Min. 1.07 51 52 53 54 55 56 58 59 61 62 64 65 66 67 68 69 70 71 72 73 75 76 77 78 80 82 84 CRL lab. code 14 ARO: Food and Residues ARO: Food and Residues

Results MPA in Batch B. Sample Batch B 2,00 ppb Std CV% ppb 1,80 Av1 1,60 CRL concentration: Av2 1,40 1.00 CRL value 1,20 conc. 1,00 Mean all laboratories (n=28) Trimmean 0,80 0.97 0.23 24 Median 1.00 2*horwitz 0,60 0,40 Mean all laboratories day 1 (n=28) 2*horwitz 0,20 1.01 0.28 28 Max. 2.00 0,00 51 52 53 54 55 56 58 59 61 62 64 65 66 67 68 69 70 71 72 73 75 76 77 78 80 82 84 CRL Mean all laboratories day 2 (n=24) lab. code 0.94 0.23 24 Min. 0.39 15 ARO: Food and Residues ARO: Food and Residues

Confirmation Gestagens in porcine kidney fat. result, expressed as a percent of all the Number of Labs performing a positiv 90 80 70 participants. 60 50 Confirmed Not confirmed 40 30 20 10 0 Batch A Batch B Batches 16 ARO: Food and Residues ARO: Food and Residues

Summary of the total number of results. 0 ≤ ⎜ Z ⎜≤ 1 1 ≤ ⎜ Z ⎜≤ 2 ⎜ Z ⎜ > 2 Sample Number of Analysed No Outlier code participants results Batch A 28 27 1 0 20 7 0 Batch B 28 28 0 0 26 2 0 Batch C 28 27 1 0 - - - Sample Min. Max. Mean Median s.d. CV% code Batch A 1.07 6.00 3.59 3.88 1.13 32 Batch B 0.39 2.00 0.97 1.00 0.23 24 17 ARO: Food and Residues ARO: Food and Residues

Evaluation of results from individual samples in the proficiency test (A). Batch A. • The overall average value (mean of means) obtained during the proficiency test was 3.59 ppb (S.D. 1.13), with no observed difference between day one and day 2. Twenty four participants had mean values above 2.0 (range 2.05 – 5.50) and 3 participants had mean values below 2.0 (range 1.16 – 1.78). • Twenty-four laboratories were able to confirm residues MPA according to the EU criteria, (Commission Decision 2002/657/EC). Four laboratories did not send us results about the confirmation of MPA. 18 ARO: Food and Residues ARO: Food and Residues

Evaluation of results from individual samples in the proficiency test (B). Batch B. • The overall average value (mean of means) obtained during the proficiency test was 0.97 ppb (S.D. 0.23), with no observed difference between day one and day 2. In view of the mean of means, there is a good agreement with the expected value. • Fourteen participants had mean values above 1.0 (range 1.00 – 1.38) and 14 participants had mean values below 1.0 (range 0.60 – 0.99). • Twenty-two laboratories were able to confirm MPA according to the EU criteria, (Commission Decision 2002/657/EC). Four laboratories did not send us results about the confirmation of MAP. 19 ARO: Food and Residues ARO: Food and Residues

Evaluation of results from individual samples in the proficiency test (C). Batch C • This sample was focused on a concentration of <0.5 ppb MPA. There were no false-positive results. • In view of the mean of means, there is a good agreement with the expected value. 20 ARO: Food and Residues ARO: Food and Residues

Scores individual labs. Graphical representation 16 14 12 Frequentie 10 8 6 4 2 0 5 7 6 8 9 scores 21 ARO: Food and Residues ARO: Food and Residues

Conclusion. •In total 30 laboratories registered as participant for the test and 28 laboratories reported their results. A limited number of participants (24 labs) was able to quantify and confirm MPA in porcine kidney fat. • 21 laboratories reported excellent results (score 8 - 9) and 6 laboratories reported good results (score 6 -7). •Only one laboratory report unsatisfactory results (scores 5). •Most of the laboratories demonstrated to be able to confirm and to quantify MPA, only four laboratories did not perform confirmatory analyses. 22 ARO: Food and Residues ARO: Food and Residues

Compared results obtained by LCMS versus GCMS. Batch A. (ppb) GC-MS LC-MSMS average 2.69 4.00 STD 0.9 0.8 CV% 34 21 N 6 17 1. Most of the participants used for detection LCMS or LCMS/MS. 2. Only six laboratories sent us results obtained by GCMS/MS. 3. The results obtained by GCMS were lower then the expected value. 23 ARO: Food and Residues ARO: Food and Residues

ARO: Food and Residues ARO: Food and Residues Overall: Excellent result! 24

Overview of the proficiency test “Thyreostatics in porcine samples of urine” November 2008 25 ARO: Food and Residues ARO: Food and Residues

Preparation of sample materials: Excretion pattern Animal 3454 3500 6000 3000 5000 Concentration (ppb 2500 4000 2000 3000 1500 2000 1000 1000 500 0 0 2005M17702005M17712005M17722005M17732005M17752005M17762005M1777 Sample nr. Tapazol-IBBR Methylthiouracil-IBBR 26 ARO: Food and Residues ARO: Food and Residues

Materials distributed • Batch A. High concentration (50 ± 10 ppb) of MTU and Tapazol. • Batch B. Low concentration (10 ± 5 ppb) of MTU and Tapazol. • Batch C. Blank samples. 27 ARO: Food and Residues ARO: Food and Residues