EVALUATION OF PROFICIENCY TESTING PROGRAM, SETTING PRIORITIES FOR THE FUTURE.

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1 Click to edit Master title style EVALUATION OF PROFICIENCY TESTING PROGRAM, SETTING PRIORITIES FOR THE FUTURE. Saskia S. Sterk Henk A. Herbold, Marco H. Blokland October 2005.

2 Introduction. One of the tasks of the CRL is to prepare testor reference materials for proficiency tests and Quality Control in NRLs.

3 Preparation of the samples 1. Animale experiment Animal Sciences Group Edelhertweg PH Lelystad the Netherlands

4 Preparation of the samples 2. Freeze-drying proces

5 Preparation of the samples 3. Quality control of the samples. Excretion study of a treated animal Homogeneity study lyophilised samples Influence of the freeze-drying proces Fast stability study

6 Overview of the subjects. 1. Evaluation of the results, proficiency testing program Overview of the current proficiency tests. 3. Overview of the proficiency tests in the near future.

7 (1) Evaluation of the results, proficiency testing program Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report Metabolism and analytical methodology for 17ßnorclostebol. Report

8 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report α-methyl-17ß-hydroxy-androsta-1,4-diene-3-one

9 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report

10 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Methyl-Boldenone is rapidly metabolised in cattle. Mass spectrum of 17α-methyl-androstene-3x,16x,17x-triol (x=α, ß). Measured mass Element composition C 29 H 56 O 3 Si 3 C 9 H 22 O 2 Si 2 C 10 H 23 O 2 Si 2 Exact mass

11 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Due to the lack of the reference standard of the metabolites, the proficiency test is focused on 1.4- androstdiene-17α-methyl-17ß-ol-3-one (= Methyl-Boldenone). Batch A (2004M2451). A sample with a concentration of ± 0.5 ppb for Methyl-Boldenone. Blank samples of urine were mixed together with incurred material in the proportion of 1440 ml to 60 ml. Batch B (2004M2452). A sample with a concentration of ± 0.2 ppb for Methyl-Boldenone. Blank samples of urine were mixed together with incurred material in the proportion of 1477 ml to 23 ml. Batch C (2004M2453).The purpose was to prepare blank samples of urine.

12 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Results Methyl-Boldenone (2004M2451 Batch A).

13 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Results Methyl-Boldenone (2004M2451 Batch A) Consensus value. With the intention to calculate a consensus value of each batch, we make a selection amongst the results. The selection is on the basis of the expected mean concentration of 0,49 ± 0,13 ppb (n=6)

14 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Sample 2004M2451 (Batch A) 9,00 Av1 7,00 Av2 5,00 Concensus value conc. 3,00 Trimmean 1,00 2*horwitz -1,00 2*horwitz -3, lab. code

15 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Results Methyl-Boldenone (2004M2452 Batch B)

16 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Confirmatory analysis

17 Results RIVM/CRL proficiency test Methyl-Boldenone in lyophilized bovine urine. Report Evaluation and discussion. Sample 2004M2451, the variations between the participant results are 0.10 till 2.51 µg/l. Five laboratories reported results corresponding to the expected concentration 0,49 ± 0,13 ppb. Sample 2004M2452 the variations are 0.14 till 1.44 µg/l. Six laboratories reported results corresponding to the expected concentration 0.26 ± 0.14 ppb. Three labs used LC-MS/MS as a technique and generated an outlier and no results.

18 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report α-Trenbolone is the major metabolite found in urine, of the anabolic steroid 17β-Trenbolone-acetate, usually indicated as Trenbolone.

19 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report Batch A (2004M2496). A mix of urine sample with a concentration of 5 (± 1) ppb for 17α-Trenbolone. Therefore blank samples of urine were mixed together with incurred material. Batch B (2004M2497). Blank samples of urine.

20 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report

21 Major steps within each procedure.

22 Major steps within each procedure.

23 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report Results Trenbolone (2004M2496 Batch A).

24 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report Consensus value. With the intention to calculate a consensus value of each batch, we make a selection amongst the results. The selection is on the basis of the values with initially IZI scores < 1. The reference value for the mass concentration 17α-trenbolone in the sample coded 2004M2496 is 4.4 μg/l, with an estimated uncertainty of 0.8 μg/l.

25 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report Sample 2004M2496 9,00 Av1 7,00 Av2 5,00 CRL value conc. 3,00 Trimmean 2*horwitz 1,00 2*horwitz -1,00 Reference value -3, lab. code

26 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report Overview of analytical results of all laboratories (μg/l) Summary of the total number of results

27 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report Overview results for each participant

28 Results RIVM/CRL proficiency test Trenbolone in lyophilized bovine urine. Report Discussion and evaluation of the results Member State representation Number of EU Member states 25 Number of responsible NRLs 24 Number of reports 20 Number of reporting Member States 17 Number of Member States with acceptable performance in at least one lab 15

29 Metabolism and analytical methodology for 17ßnorclostebol. Report Molecular structure of norclostebol-propionate and metabolites. 4 -chloro-17α-hydroxy-19-norandrostane-3-one 4-chloro-3-hydroxy-19-norandrostane-17-one

30 Metabolism and analytical methodology for 17ßnorclostebol. Report Metabolism study. O Si Mass-spectrum of 17ß-norclostebol O Si Cl Mass-spectrum of 17α-norclostebol (metabolite I). O O Si Si Cl O Si O Si O Si Cl O Si Cl

31 Metabolism and analytical methodology for 17ßnorclostebol. Report chloro-17α-hydroxy-19-norandrostane-3-one (Urinary metabolite II) Spectrum of the synthesized reference compound 4-chloro-17ß-hydroxy-19-norandrostane-3-one (Molecular ion m/z 364).

32 Metabolism and analytical methodology for 17ßnorclostebol. Report For residue control the metabolite I and II seem to be the most appropriate. Unfortunately, for both compounds, reference compounds are not available. In practise the situation is as follows: Metabolite I: 17α-norclostebol Reference I: 17β-norclostebol Metabolite I: 4-chloro-17α-hydroxy-19-norandrostane-3-one Reference II: 4-chloro-17ß-hydroxy-19-norandrostane-3-one

33 Metabolism and analytical methodology for 17ßnorclostebol. Report Reference I, 17ß-Norclostebol ret. time 9.35 min. (m/z 452, 454). Metabolite I, 17α-Norclostebol in urine ret. time 9.13 min. (m/z 452, 454).

34 Metabolism and analytical methodology for 17ßnorclostebol. Report Reference II, 4-chloro-17ß-hydroxy-19-norandrostane-3-one, ret. time 8.90 min. (m/z 366, 364). Metabolite II, 4-chloro-17α-hydroxy-19-norandrostane-3-one in urine, ret. time 8.75 min. (m/z 366, 364).

35 Metabolism and analytical methodology for 17ßnorclostebol. Report Excretion curve Estimated Concentration (ppb) Days Norclostebol Metabolite Excretion curve of 17α-norclostebol (MI, blue colour) and the metabolite 4-chloro-17α-hydroxy-19-norandrostane-3-one (MII, red colour).

36 Metabolism and analytical methodology for 17ßnorclostebol. Report Batch A (2004M0781). A urine sample with a high concentration of 17αnorclostebol. Batch B (2004M0782). A urine sample with a middle concentration of 17αnorclostebol. Batch C (2004M0783).A urine sample with a high concentration of the metabolite 4-chloro-17α-hydroxy-19-norandrostane-3-one. Batch D (2004M0784).A urine sample with a middle concentration of the metabolite 4-chloro-17α-hydroxy-19-norandrostane-3-one. Batch E (2002M0889). Blank samples of urine.

37 Metabolism and analytical methodology for 17ßnorclostebol. Report

38 Metabolism and analytical methodology for 17ßnorclostebol. Report Results 17α-norclostebol and metabolite II, (ppb)

39 Metabolism and analytical methodology for 17ßnorclostebol. Report Results 17α-norclostebol and metabolite II

40 Metabolism and analytical methodology for 17ßnorclostebol. Report Confirmation

41 Metabolism and analytical methodology for 17ßnorclostebol. Report Discussion and evaluation of the results In total 10 labs participated in this study, 4 of which submitted a report. 2 participants reported results for both compounds (14 and 25). Participant 14 was able to detect and confirm the analytes in all samples. 1 participant analysed all samples for 17α-norclostebol (20). Quantitative results were similar to other values reported, but full confirmation was not possible. 1 participant analysed all samples for metabolite II (33) and was able to confirm the identity. The compound 17α-norclostebol and 4-chloro-17-hydroxy-19- norandrostane-3-one, were not analyzed by a large number of labs.

42 (2) Overview of current proficiency tests. Results RIVM/CRL proficiency test DES in lyophilized bovine urine. (report available the end of 2005) RIVM/CRL proficiency test Ethynyloestradiol in lyophilized bovine urine. (Current test) RIVM/CRL proficiency test oestradiol in lyophilized bovine urine and plasma. (2005/2006) Metabolism and analytical methodology for 19Nor-5 androstene 3,17-dione. (A study among NRLs) (2005/2006)

43 (3) Overview of proficiency tests in the near future. Animal experiment with pigs. Pigs are treated with: Trenbolonacetate Medroxyprogesterone (MPA) Thyreostatica (Tapazol, MTU) Organisation: ringtests among NRLs, CRLs Metabolism studies among NRLs Preparation of reference materials

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