RIVM document Results RIVM/CRL proficiency test: Nortestosterone in lyophilised bovine urine.

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2 RIVM document Results RIVM/CRL proficiency test: 17α -/17β- Boldenone and 17α -/17 β- Nortestosterone in lyophilised bovine urine. November 2001 This study was performed within the task of the European Union Communities Reference Laboratory (CRL) for Residues.

3 EU Community Reference Laboratory National Institute of Public Health and the Environment Laboratory for Residue Analysis P.O. Box BA Bilthoven The Netherlands tel. ** fax. ** crl.aro@rivm.nl 2

4 3 Preface. This report describes the results of the second proficiency test organised by the EU Community Reference Laboratory for residues at RIVM, Bilthoven, The Netherlands. Proficiency testing is one of the key activities of the EU Community Reference Laboratories. On several occasions, representatives of National Reference Laboratories expressed their interest and indicated the need for reference materials, reference standards and proficiency testing. Consequently, RIVM/CRL is running a continuous series of animal experiments in order to obtain incurred biological samples. These materials are used as sample materials in proficiency tests and as source materials for the development of reference materials. The CRL acknowledges the contribution of all participants and thanks the European Commission for its financial contribution. Henk A. Herbold Saskia S. Sterk Rainer W. Stephany Leendert A. van Ginkel The results of your laboratory are included under number:

5 4 Table of Contents Page Summary 5 Introduction 7 List of participants 8 Sample materials 9 Verification analyses 14 Results proficiency test 17 Discussion and evaluation 61 References 64

6 5 Summary This report describes the results of the second proficiency test organised by the EU Community Reference Laboratory for residues at RIVM, Bilthoven, The Netherlands. In total 23 laboratories registered as participant, including the National Reference Laboratories of all EU Member States, one National Reference Laboratory of a candidate Member State as well as a number of field laboratories. Finally, 17 laboratories reported their results. The proficiency test focussed on residue control for 19-Nortestosterone and Boldenone and was based on lyophilised samples of bovine urine incurred with the major metabolites and spiked with the parent compounds. For Nortestosterone, three Member States did not demonstrate their capability for performing adequate residue control, neither through their National Reference Laboratory nor through a Routine or Field Laboratory. One Member State does not perform testing for the relevant metabolite. For Boldenone it is concluded that only a relative small number of Member States at this moment perform residue control. In view of the current uncertainties with respect to the possible natural occurrence of 17α- and 17β-Boldenone it is suggested to give a relative low priority to the incorporation of Boldenone in National Residue Control programmes, pending a stronger scientific basis.

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8 7 Introduction One of the tasks of the CRL is to prepare test- and/or reference materials and to organise proficiency tests for compounds within its mandate. Proficiency tests assist National Reference Laboratories (NRLs) in maintaining Quality Assurance and Quality Control (QA/QC) programmes. Additionally, information is obtained with respect to the analytical possibilities within individual laboratories. This study was organised in the spring of 2000 by the Community Reference Laboratory (CRL) for Hormonal Anabolic Compounds, Mycotoxins and Sedatives, Bilthoven, The Netherlands. The purpose of this study was to evaluate current analytical methodology for the detection and determination of low levels of 17α- and 17ß- Boldenone (dehydrotestosterone) and 17α- and 17ß- Nortestosterone in samples of lyophilised bovine urine. At the same time the development of reference materials for these compounds was foreseen. An important requirement for proficiency tests is to use incurred material in order to create conditions which are as similar as possible to real-life samples. Therefor, material obtained from a treated young female cow was used for this proficiency test. CH 3 OH CH 3 OH H H H H O H H O H H 17α-Nortestosterone cas no: β-Nortestosterone cas no: CH 3 OH CH 3 OH CH 3 H CH 3 H O H H O H H 17α-Boldenone cas no: β-Boldenone cas no: Figure 1: Molecule structures of the analytes.

9 8 List of participants proficiency test in random order. Rikilt (NRC) University of Ghent Bornsesteeg 45 Dept. Lab. chemical analysis 6708PD Wageningen Salisburylaan 133 the Netherlands 9820 Merelbeke Belgium Min. de l Agriculture et de la Pêche Veterinary Sciences Division Ecole Nationale Vétérinaire Dept. Biochemistry Route de Gachet BP Stoney road, Stormont F Nantes Cedex 03 Belfast BT4 France Northern Ireland UK Min. van Middenstand en Landbouw Rijksontledingslaboratorium National Food Administration Department Residues Dept. Chemistry div.1 Braemkasteelstraat 59 Strandbodgatan Gentbrugge S Uppsala Belgium Sweden State Laboratory Abbotstown Cent. Nacional de Alimentation Abbotstown, Castleknock Instituto de Salud Carlos 111 IRL-Dublim 15 Unidad Zoosanitarios Ireland CTRA Majadahonda-Pozuelo E Madrid Majadahonda España. National Veterinary and food Lab. Nacional de Investigacäo Research Institute Veterinária Dept. Chemistry Estrada de Benefica 701 Hämeentie 57 P Lisboa. FIN Helsinki Portugal Finland State General Laboratory Centraal Laboratorium RVV Veterinary Drug Residue Ministerie LNV 44 Kimonos ST. P.O.Box 144 Nicosia AC Wageningen Nicosia Cyprus. the Netherlands

10 9 Danish Veterinary and Food Centre D economie Rurale Administration. Laboratoire d Hormonologie Veterinary Drug Residues Rue du Carmel 1 Sondervang 4 B-6900 Marloie DK-4100 Ringsted Belgium Denmark TNO-Voeding WIV - Louis Pasteur Pharmacology Juliëtte Wytsmanstraat 14 Utrechtseweg 48 B-1050 Brussel 3700AJ Zeist Belgium the Netherlands BGVV - Bundesinstitut für Ministry of Agriculture Gesundheitlichen Verbraucher schutz Veterinary Laboratory of und Veterinär medizin. Serres. Thielallee 88/92 Terma Omonoias D Berlin GR Germany. Greece. Istituto Superiore di Sanitá Bundesanstalt für Laboratorio di Medicina Veterinaria Tierseuchenbekämpfung. Viale Regina Elena 299 Abteilung Hormonanalytik und Roma ß-Agonisten Italia. Robert Koch-Gasse 17 A-2340 Mödling Österreich LGC Laboratory for the Government Chemist Queens Road TW11 OLY Teddington Middlesex United Kingdom Dr. L. Willems-Instituut Universitaire Campus, Geb. C B-3590 Diepenbeek Belgium National Institute of Public Health and the Environment P.O. Box BA Bilthoven The Netherlands

11 10 Samples materials. Production. The sample materials supplied during this proficiency test were obtained from a young (12 month) female bovine animal. This animal was treated by intramuscular injection with nortestosterone-fenylproprionate (Anabolin forte, Alfasan, REG NL 4423 UDA, lot , 200 mg/4 ml) on day 0 and day 7 and with Boldenoneundecylenate (Boldane, Novartis, REG NL 5268 UDA, lot , 200 mg/8 ml) on day 28 and day 35). Afterwards, the animal was treated with dexamethasonefenylproprionate (Dexafort, Intervet, REG NL 2894 UDA, lot 28084B, 30 mg/10ml) on day 56 and 63. Samples of urine were collected on day 1, 2, 3, 7, 7, 8, 10, 15, 28, 29, 30, 31, 33, 35, 36, 38, 43 and 56. All samples of urine were stored in the freezer at 20 o C until analysis. All samples of urine obtained were analysed according to ARO procedures 113 and 401, both based on gas chromatography mass spectrometry (GC-MS). Table 1 summarizes the results. Selected samples were blended with blank bovine urine to obtain target concentrations. Each sample was numbered with a unique number generated with the management information system (ARO-MIS). Table 1. Excretion of 17α- and 17β-Nortestosterone and 17α- and 17β-Boldenone after treatment of a female bovine animal (age 1 year) 1 Day Sample id (ARO number) 17β-NT (µg/l) 17α-NT (µg/l) 17β-Bol (µg/l) 17α-Bol (µg/l) M1343 < 0.1 < 0.1 < 0.1 < 0.1 Treatment with nortestosterone fenylproprionate 0 99M < 0.1 < M < 0.1 < M < 0.1 < M < 0.1 < M < 0.1 < M1349 < < 0.1 < 0.1 Treatment with nortestosterone fenylproprionate 8 99M < 0.1 < M < 0.1 < M < 0.1 < M1353 < < 0.1 < 0.1 Treatment with Boldenone undecylenate 28 99M M M M M M Treatment with Boldenone undecylenate 36 99M1360 < Study ID_DLO April 1999

12 11 Day Sample id (ARO number) 17β-NT (µg/l) 17α-NT (µg/l) 17β-Bol (µg/l) 17α-Bol (µg/l) 38 99M1361 < M M1363 < Treatment with dexamethasone fenylproprionate 56 99M1364 < M1365 < M1366 < M1367 < M1368 < M1369 < Treatment with dexamethasone fenylproprionate 64 99M1370 < M1371 < M1372 < M1373 < Graphical representations are given in Figure ß-NT 17a-NT 17ß-Bol 17a-Bol Figure 2. Diagram of the excretion of 17α-, 17β-Nortestosterone and 17α-, 17β- Boldenone, after treatment.

13 12 Table 2. Samples prepared for this study. Batch Sample number 2001M1378 blank urine 99M1359* 99M1360* Cap colour ID-DLO Batch M ml 300 ml Blue Batch M ml 1200 ml Green Batch M ml 3000 ml Lila Batch M ml Red 1 Description preparation - Batch 1 (2000M1842). The purpose was to prepare a sample of urine sample with a low concentration (approximately 0.5 µg/l 17α-, 17β Nortestosterone and 17α-, 17β Boldenone. The blank urine was blended with incurred material in amounts of 5700 and 300 ml respectively. Further, this mixture was fortified with 2500 ng of 17ß-Boldenone and 2500 ng of 17 ß- Nortestosterone. 2 Description preparation - Batch 2 (2000M1843). The purpose was to prepare a sample of urine sample with a intermediate concentration (range >2 till < 5 µg/l 17α-, 17β Nortestosterone and 17α-,17β- Boldenone. The blank urine was blended with incurred material in amounts of 4800 and 1200 ml respectively. Further, this mixture was fortified with 10 µg of 17ß-Boldenone and 10 µg of 17ß-Nortestosterone. 3 Description preparation - Batch 3 (2000M1844). The purpose was to prepare a mix of urine sample with a high concentration (approximately 5 µg/l 17α-, 17β Nortestosterone and 17α-,17β-Boldenone. The blank urine was blended with incurred material in amounts of 3000 and 3000 ml respectively. Further, this mixture was fortified with 24 µg of 17ß-Boldenone and 24 µg of 17ß-Nortestosterone. *Incurred material see Table 2.

14 13 Before lyophilisation the four individual batches were filtered through a 0.2 µm filter. Sample aliquots of 10 ml were filled in brown glass vials. In order to check the variation of this procedure, a set of 6 vials of each batch was selected randomly and weighed before and after filling, to calculate the net filled weight and the coefficient of variation (CV) (Table 3). Table 3. Weights in grams of 6 control-vials before and after filling. Batch 1 Number Weight vial Weight vial + urine Weight Lyophilised sample (vial + urine) The loss of weight Dry weight STD CV% Batch 2 Number Weight vial Weight vial + urine Weight Lyophilised sample (vial + urine) The loss of weight Dry weight STD CV% Batch 3 Number Weight vial Weight vial + urine Weight Lyophilised sample (vial + urine) The loss of weight Dry weight STD CV% Batch 4 Number Weight vial Weight vial + urine Weight Lyophilised sample (vial + urine) The loss of weight Dry weight STD CV%

15 14 After filling, the vials were loosely fitted with a rubber stopper and lyophilised. After lyophilisation, the samples were labelled with a sample-number (Table 2), capped and stored at -70 o C. After verification analyses, in order to check the homogeneity, the samples were ready for distribution. Participants received 6 vials of the three batches containing incurred residues and 2 vials containing the blank. Lyophilisation was performed with a Christ Epsilon 2 freeze-dryer. The lyophilisation process was started when the temperature of the sample material was below the eutectic temperature. When the process was finished, the chamber was filled with nitrogen and the vials were closed automatically. The vials were fitted with an aluminium cap and labelled. Verification analyses Homogeneity and reproducibility. From each batch, a set of 6 vials was selected randomly before and after lyophilisation and analysed according to ARO SOP 401 to check the homogeneity, the content of the vials and the influence of the lyophilisation process on the average mass concentration. The concentrations were measured on two different days. The average values are listed in Table 4, 5 and 6. Table 4. Batch 1, concentrations in µg/l before and after lyophilisation (N = 2) 2000M1842 α-nt ß-NT α-bol ß-Bol Average STD CV% Italics: after lyophilisation. Table 5. Batch 2, concentration in µg/l before and after lyophilisation (N = 2) 2000M1843 α-nt ß-NT α-bol ß-Bol Average STD CV% Italics: after lyophilisation.

16 15 Table 6. Batch 3, concentration in µg/l before and after lyophilisation (N = 2) 2000M1844 α-nt ß-NT α-bol ß-Bol Average STD CV% Italics: after lyophilisation. Remarks. Comparison of the average values before and after lyophilisation, shows a relative large difference (>50%) in concentration for 17α-NT in batch 1. For all analytes there is a small negative trend in concentration observed after lyophilisation. Comparison of the average values before and after lyophilisation, shows a relative large difference (>50%) in concentration for 17α-Boldenone in batch 3. Comparison of the CV values with the CV values of 17α- and 17β NT and 17αand 17β-Boldenone in SOP 401 (RIVM report ), shows in case of α- Boldenone a relative large difference (>50%), especial in batch 1 and 3. The quantitative analyse of 17α- Boldenone in samples of urine by GC-MSD with an acceptable CV (< 20%) needs future optimization. Fast stability study. Stability experiments were performed to verify the stability of the materials, e.g. during shipment. From each batch 15 vials were stored in the dark at 37 o C for a period of 2, 4, 6, 8, 10 weeks (2 vials per period). After each period the units were analysed. The average values of two parallel determinations of each period are listed in Table 7. Table 7, Stability study at 37 o C (Concentrations in µg/l). 14-days 28-days 42-days 56-days 70-days CV% Batch 1 α-nt 1,4 1,4 1,4 1,6 1,5 6 β-nt 0,2 0,2 0,1 0,3 0,2 35 α-bol 4,5 4,3 4,6 4,8 5,7 11 β-bol 0,4 0,4 0,5 0,5 0,4 12 Batch 2 α-nt 4,0 4,5 3,8 3,8 4,0 7 β-nt 2,1 2,3 1,7 1,6 1,7 16 α-bol 13,2 13,5 20,2 17,3 18,7 19 β-bol 3,0 2,9 2,5 2,3 2,3 13

17 16 14-days 28-days 42-days 56-days 70-days CV% Batch 3 α-nt 10,0 10,8 9,2 11,3 10,1 8 β-nt 4,8 5,0 4,1 4,9 3,9 11 α-bol 30,1 36,6 37,1 45,2 40,8 15 β-bol 7,1 6,1 6,9 6,8 5,8 9 The stability is acceptable, with no significant differences during the period of testing. In case of 17β-NT in batch 1 the CV is >20%, considered acceptable in view of the fact that this value is close to the limit of detection. Graphical representations are given in Figures 3 to 5. Figure 3, Stability study Batch 1. Batch 1 6 ppb alpha-nt beta - N T alpha - Bold beta - Bold Storage days Figure 4, Stability study Batch 2. Batch 2 ppb alpha-nt beta - NT alpha - Bold beta - Bold Storage days Figure 5, Stability study Batch 3. Batch 3 ppb alpha-nt beta - NT alpha - Bold beta - Bold Storage days

18 17 Results proficiency test In September 2000, sample materials were distributed to 23 laboratories. Six laboratories did not submit any results. Of the remaining 17 laboratories eight reported results for all analytes. The other laboratories submitted results for two or three analytes, The last results were received in July Table 8. (Nr = no results, + = analysed, - = not analysed ). Labcode 17α- Nortestosterone 17β- Nortestosterone 17α- Boldenone 17β- Boldenone 0 (CRL) Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr Nr 22 Nr Nr Nr Nr Total Table 9. Number of reporting participants. α-nt β-nt α-bold β-bold Remarks Analysed Number of participants that submitted results. Confirmed Number of participants that submitted confirmatory results. Not analysed Number of participants that didn t analyse for the analyte. No results Number of participants that didn t send us any results.

19 18 Analytical steps in the procedure used by the participants. All laboratories used a combination of glucuronidase/sulfatase for enzymatic deconjugation of analytes. The analytes were extracted by solid phase extraction (14 labs) or, in a few cases by liquid/liquid extraction (2 labs). All laboratories used a preparative HPLC- technique. In one laboratory a combination of three extraction techniques was used. One laboratory did not submit any information about the cleanup procedure used. After derivatisation of the purified extracts, gas chromatography coupled with a mass spectrometric (GC-MS) detection was applied to screen and to confirm the samples for the presence of residues of 17α - 17β Nortestosterone and 17α - 17β Boldenone in all cases but 1. One laboratory used LC-MS for detection. Table 10 summarise the methods and their characteristics. Abbreviations used GC-MS EI LC-MS GC-MS APCI NT SPE IAC HPLC Gas Chromatography - mass spectrometry Electron Impact Liquid Chromatography - mass spectrometry Liquid Chromatography - Mass Spectrometry Atmospheric Pressure Chemical Ionisation 19 Nortestosterone Solid Phase Extraction Immunoaffinity Chromatography High Performance Pressure Chromatography

20 19 Table 10. Summarises the major steps within each procedure. Lab. code. Methods Technique Analyte Internal standards 0 GCMS-EI ß-NT-d3 CRL Hydrolysis glucuronidase/sulfatase SPE on C18/NH 2 HPLC (coupled column) Derivatisation HFBA 1 Hydrolysis glucuronidase/sulfatase SPE on C18/NH 2 HPLC fraction Derivatisation MSTFA ++ 2 Hydrolysis glucoronidase/sulfatase LLE xtraction HPLC fraction Derivatisation MSTFA ++ 3 Hydrolysis glucuronidase/sulfatase SPE on C18/Si HPLC fraction Derivatisation MSTFA ++ 4 Hydrolysis glucuronidase/sulfatase SPE on C18/NH 2 HPLC fraction Derivatisation HFAA GC-hrMS for confirmation 5 Hydrolysis glucuronidase/sulfatase HPLC fraction Derivatisation HFBA 7 Hydrolysis glucuronidase/sulfatase SPE, Derivatisation HFBA 8 Hydrolysis glucuronidase/sulfatase SPE on C18/NH 2 /Si Derivatisation MOX 9 Hydrolysis glucuronidase/sulfatase SPE on C18 LLE, HPLC fraction Derivatisation BSTFA/TMCS GCMS-EI GCMS-EI GCMS-EI LC-MS- MS Methylbol-d3 GCMS- MS GCMS-EI GCMS- EI/CI GCMS-EI α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. ß-Bol-d3 ß-NT-d3 methylbold3 Limit of detection (ppb) ß-NT-d not available 0.73 ß-NT-d3 Methylbol-d3 Estradiol-d3 ß-NT-d None ß-NT-d not available not available ß-NT-d Dienestrol

21 20 Lab. code. Methods Technique Analyte Internal standards 11 Hydrolysis GCMS-EI α - NT ß-NT-d3 glucuronidase/sulfatase SPE on C18, HPLC α - Bold. Estradiol-d3 fraction β - Bold. Derivatisation HFBA 13 Hydrolysis GCMS- α - NT glucuronidase/sulfatase NCI (J. Chrom. A α - Bold. 867(2000), ) β - Bold. Derivatisation HFBA 15 Hydrolysis glucuronidase/sulfatase SPE on Oasis HPLC fraction Derivatisation MSTFA Hydrolysis glucuronidase/sulfatase SPE HPLC 17 Hydrolysis glucuronidase/sulfatase SPE on C18 HPLC fraction Derivatisation HFBA 18 Hydrolysis glucuronidase/sulfatase IAC clean-up Derivatisation BSTFA 19 Hydrolysis glucuronidase/sulfatase SPE on Si/C18 HPLC fraction Derivatisation BSTFA 20 Hydrolysis glucuronidase/sulfatase SPE on C18 HPLC fraction Derivatisation HFBA 23 C18/Hydrolysis glucuronidase/sulfatase SPE on C18 HPLC fraction/iac Derivatisation TMS GCMS-EI HPLC EIA (βnt LCMS) GCMS-EI GCMS-EI GCMS-EI GCMS-EI GCMS-EI α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. α - NT α - Bold. β - Bold. Limit of detection (ppb) not available not available not available not available ß-NT-d ß-NT-d3 ß-NT-d3 ß-NT-d3 ß-NT-d3 ß-NT-d not available ß-NT-d not available not available ß-NT-d not available not available ß-NT-d not available not available methyltestd3 Testosterond3 0.4 not available not available not available

22 21 Evaluation of Results Results were evaluated in terms of Z-scores. In order to avoid excessive values for the variability in the mean observed values, extremely deviating results were excluded (outliers according to Grubbs test). Also false negative results and results without quantification were not included in the overall evaluation. For the calculation of the Z-score, two values must be determined: target standard deviation and the mean value of all laboratories per sample. The Z-score was calculated for each laboratory according to the equation given below: Z score = Xlab Xmean Q Xlab Xmean Q : mean value of the laboratory per sample : mean value of all laboratories per sample : standard deviation for the mean value of all laboratories. The advantage of the Z-score determination is that it provides a standardised value allowing to compare the results both within one interlaboratory study and between different interlaboratory studies irrespective of the concentration of the analyte. In the case of a normal distribution, the probability of the absolute value of Z not exceeding the value 2 is approximately 95% [5]. It is therefore sensible to establish the value 2 as a quality limit for the underlying measurements. Thus a laboratory with a Z value of <2 is generally regarded as having performed satisfactory, a Z score between 2 and 3 questionable, and Z scores >3 are unacceptable. The individual values listed in the tables below are coloured green in case the absolute Z score is < 1, blue in case the score > 1, but does not exceed 2 and red in case of an absolute Z score >2 or in case of a false positive or false negative result.

23 22 Evaluation sample 2000M1842 (batch 1) for 17α-Nortestosterone. Target concentration (estimated): 0,7 µg 17α-Nortestosterone /l Number of participants: 15 Number of results evaluated: 13 Number of sets excluded from the evaluation 2 The individual results are summarised in Table 11. Figure 6 gives a graphical representation of the individual results and the distribution of Z-scores. Table 11. Results (µg/l) for 17-Nortestosterone: Batch 1, sample 2000M1842. Lab.no Day 1 Day 2 Mean(*) s.d. CV% Z-score Remarks CRL (0) 0,62 0,65 0,64 0,02 3,3-1,23 1 1,80 2,00 1,90 0,14 7,4 0,76 2 <LOD 0,16 0,16-1, ,00 1,60 1,80 0,28 15,7 0,61 4 1,79 2,38 2,09 0,42 20,0 1,05 5 0,70 1,40 1,05 0,49 47,1-0,58 7 2,50 2,20 2,35 0,21 9,0 1,47 8 2,00 < LOD 2,00 0, ,70 1,90 1,80 0,14 7,9 0, ,49 1,50 1,50 0,01 0,5 0, ,00 4,90 4,45 0,64 14,3 Outlier 15 <LOD <LOD < LOD Fn 16 1,50 1,20 1,35 0,21 15,7-0, ,00 1,10 1,05 0,07 6,7-0, ,28 1,70 1,49 0,30 19,9 0, ,40 0,90 0,65 0,35 54,4-1,20 Mean 1,44±0,62 1,44±0,63 1,42±0,61 (*) If only day 1 or 2 is performed, this concentration is used as mean value. (LOD: Limit of Detection, Fn: False negative). (Mean: except Outlier)

24 23 Figure 6. Laboratory results for Batch 1, 17α-Nortestosterone, individual values (above) and Z-scores (below). 3,00 α-nt batch 1 (Low concentration) 2,50 2,00 ppb 1,50 1,00 0,50 0, Labcodes Mean Experiments Mean Labs CRL +std -std Z-score α-nt (Low concentration) Z-score 2 1,5 1 0,5 0-0,5-1 -1,5-2 -2, Labcodes

25 24 Evaluation sample 2000M1843 (batch 2) for 17α-Nortestosterone. Target concentration (estimated): 3,2 µg 17α-Nortestosterone /l Number of participants: 15 Number of results evaluated: 14 Number of sets excluded from the evaluation 1 The individual results are summarised in Table 12. Figure 7 gives a graphical representation of the individual results and the distribution of Z-scores Table 13. Results (µg/l) for 17α-Nortestosterone. Batch 2, sample 2000M1843, Lab.no Exp. day 1 Exp. day 2 Mean s.d. CV% Z-score Remarks CRL (0) 3,28 3,32 3,30 0,03 0,9-0,12 1 5,20 5,10 5,15 0,07 1,4 1,54 2 1,85 4,37 3,11 1,78 57,3-0,27 3 3,30 3,40 3,35 0,07 2,1-0,08 4 3,76 4,93 4,35 0,83 19,0 0,69 5 2,10 2,70 2,40 0,42 17,7-0,83 7 4,80 4,30 4,55 0,35 7,8 0,85 8 3,60 4,80 4,20 0,85 20,2 0,58 9 3,40 3,40 3,40 0,00 0,0-0, ,01 4,02 4,02 0,01 0,2 0, ,10 8,60 9,35 1,06 11,3 Outlier 15 1,70 2,20 1,95 0,35 18,1-1, ,00 3,00 3,00 0,00 0,00 0, ,70 3,60 2,65 1,34 50,7-0, ,95 1,94 1,95 0,01 0,4-1, ,50 1,40 1,45 0,07 4,9-1,57 Mean 3,0 ± 1,2 3,5 ± 1,1 3,3 ± 1,2 (LOD: Limit of Detection, Fn: False negative).

26 25 Figure 7. Laboratory results Batch 2 (sample 2000M1843) for 17α-nortestosterone, individual values (above) and Z-scores (below). 7,00 α-nt batch 1 (medium concentration) 6,00 5,00 ppb 4,00 3,00 2,00 1,00 0, Labcodes Mean Experiments Mean Labs crl +std -std Z-score 2,5 2 1,5 1 0,5 0-0,5-1 -1,5-2 Z-score α-nt (medium concentration) Labcodes

27 26 Evaluation sample 2000M1844 (batch 3) for 17α-Nortestosterone. Target concentration (estimated): 8,0 µg 17α-Nortestosterone /l Number of participants: 15 Number of results evaluated: 14 Number of sets excluded from the evaluation 1 The individual results are summarised in Table 14. Figure 8 gives a graphical representation of the individual results and the distribution of Z-scores Table 13. Results (µg/l) for 17α-nortestosterone: Batch 3, sample 2000M1844. Lab.no Exp. day 1 Exp. Day 2 Mean s.d. CV% Z-score Remarks CRL (0) 8,07 8,10 8,09 0,02 0,3-0, ,8 12,5 12,65 0,21 1,7 1,28 2 0,65 2,00 1,33 0,95 72,0-1,99 3 6,10 9,30 7,70 2,26 29,4-0, ,15 14,14 12,65 2,11 16,7 1,28 5 5,70 6,40 6,05 0,49 8,2-0,62 7 8,70 13,00 10,85 3,04 28,0 0, ,50 14,10 12,30 2,55 20,7 1,18 9 9,50 8,10 8,80 0,99 11,2 0, ,03 11,45 11,24 0,30 2,6 0, ,60 25,70 23,65 2,90 12,3 Outlier 15 9,80 10,20 10,00 0,28 2,8 0, ,70 6,40 6,05 0,49 8,2-0, ,6 11,45 7,53 5,55 73,8-0, ,83 3,87 4,35 0,68 15,6-1, ,60 2,80 3,70 1,27 34,4-1,30 Mean 7,5 ± 3,4 8,9 ± 4,0 8,2 ± 3,7 (LOD: Limit of Detection, Fn: False negative).

28 27 Figure 8. Laboratory results Batch 3, 17α-Nortestosterone, individual values (above) and Z-scores (below) α-nt batch 1 (high concentration) 16,00 14,00 12,00 10,00 ppb 8,00 6,00 4,00 2,00 0, Labcodes Mean Experiments Mean Labs +std -std crl Z-score α-nt (high concentration) Z-score 1,5 1 0,5 0-0,5-1 -1,5-2 -2, Labcodes

29 28 Evaluation sample 2000M1845 (batch 4) for 17α-Nortestosterone. Target concentration (estimated): <0,1 µg 17α-Nortestosterone /l Number of participants: 15 Number of results evaluated: 15 Number of sets excluded from the evaluation 0 The individual results are summarised in Table 14. Table 14 Results (µg/l) for 17α-nortestosterone: Batch 4, sample 2000M1845. Lab.no Day 1 Day 2 Mean s.d. CV% Z-score Remarks CRL (0) <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD

30 29 Discussion results for 17α-Nortestosterone. 17α-Nortestosterone is the most interesting compound during this proficiency test. It is well documented that effective residue control for the abuse of 19-Norterstosterone by analyses of samples of urine is through the analyses for the presence of 17α- Nortestosterone, the major metabolite. Excluding the CRL in total 15 laboratories reported results. The status of these laboratories is summarised in Table 15. Table 15: Overview results in terms of category participant (N=15) EU NRL EU RFL EU-ISC NON-EU NRL Reports False negative low levels False negative higher levels Outlying quantification Adequate results NRL: RFL: ISC: NON-EU NRL: National Reference Laboratory Routine/Field Laboratory Industry Self Control Laboratory (officially part of national Residue Control Programme) National Reference Laboratory candidate EU Member State In total ten EU NRLs reported their results. Together they represent nine Member States. One Member State was represented through two RFLs. One NRL reported a false negative result. However, at the low mass concentration concerned this can be considered as a non critical error. The outlying quantification as reported by another NRL indicates a problem with standardisation but is not critical in terms of the ability of the laboratory to detect the abuse of 19-Nortestosterone. One NRL did only analyse the samples for the parent compound 17β-Nortestosterone. From a control point of view this is critical, from an analytical point of view this can be adjusted relative simply. In conclusion nine Member States reported acceptable results. Two Member States demonstrated at least to have in principle the capability of performing acceptable analytical residue control for 19-Nortestosterone. The three other Member States include Luxembourg which is represented by another Member State. The remaining two Member States until now have not demonstrated this capability.

31 30 Evaluation sample 2000M1842 (batch 1) for 17β-Nortestosterone. Target concentration (estimated): 0,42 µg 17β-Nortestosterone /l Number of participants: 16 Number of results evaluated: 9 Number of sets excluded from the evaluation 7 The individual results are summarised in Table 16. Figure 9 gives a graphical representation of the individual results and the distribution of Z-scores. Table 16. Results (µg/l) for 17β-nortestosterone: Batch 1, sample 2000M1842 Lab.no Exp. day 1 Exp. day 2 Mean(*) s.d. CV% Z-score Remarks CRL (0) 0,61 0,69 0,65 0,06 8,7 0,80 1 0,40 0,50 0,45 0,07 15,7-0,43 2 0,39 <LOD 0,39-0,80 3 <LOD <LOD <LOD Fn 4 <LOD <LOD <LOD Fn 5 0,60 0,50 0,55 0,07 12,9 0,19 7 <LOD 0,90 0,90 2,35 8 <LOD <LOD <LOD Fn 9 <LOD <LOD <LOD Fn 11 0,36 0,39 0,38 0,02 5,7-0,89 13 <LOD <LOD <LOD Fn 16 <LOD <LOD <LOD Fn 17 5,70 5,20 5,45 0,35 6,5 Outlier 18 0,49 0,58 0,54 0,06 11,9 0, ,50 0,50 0,50 0,00 0,0-0, ,34 0,35 0,35 0,01 2,0-1, ,40 0,60 0,50 0,14 28,3-0,12 0,45±0.10 0,56±0.17 0,52±0.16 (*) If only day 1 or 2 is performed, this concentration is used as mean value. (LOD: Limit of Detection, Fn: False negative).

32 31 Figure 9. Laboratory results Batch 1, 17β-Nortestosterone, individual values (above) and Z- scores (below). ppb β-nt batch 1 (Low concentration) 1,00 0,90 0,80 0,70 0,60 0,50 0,40 0,30 0,20 0,10 0, Labcodes Mean Experiments Mean Labs +std -std crl Z-score β-nt (Low concentration) Z-score 3 2,5 2 1,5 1 0,5 0-0,5-1 -1, Labcodes

33 32 Evaluation sample 2000M1843 (batch 2) for 17β-Nortestosterone. Target concentration (estimated): 1,7 µg 17β-Nortestosterone /l Number of participants: 16 Number of results evaluated: 15 Number of sets excluded from the evaluation 1 The individual results are summarised in Table 17. Figure 10 gives a graphical representation of the individual results and the distribution of Z-scores. Table 17. Results (µg/l) for 17β-nortestosterone: Batch 2, sample 2000M1843. Lab.no Exp. day 1 Exp. day 2 Mean(*) s.d. CV% Z-score Remarks CRL (0) 2,10 2,07 2,09 0,02 1,0 0,57 1 2,50 2,10 2,30 0,28 12,3 1,04 2 0,25 1,22 0,74 0,69 93,3-2,37 3 1,90 1,70 1,80 0,14 7,9-0,05 4 1,70 1,77 1,74 0,05 2,9-0,19 5 2,00 1,90 1,95 0,07 3,6 0,28 7 2,10 2,10 2,10 0,00 0,0 0,60 8 1,00 1,30 1,15 0,21 18,4-1,47 9 1,50 1,80 1,65 0,21 12,9-0, ,77 1,83 1,80 0,04 2,4-0, ,30 4,80 4,55 0,35 7,8 Outlier 16 1,90 <LOD 1,90 0, ,25 3,45 2,85 0,85 29,8 2, ,66 1,67 1,67 0,01 0,4-0, ,90 1,70 1,80 0,14 7,9-0, ,76 1,76 1,76 0,00 0,0-0, ,90 1,90 1,90 0,00 0,0 0,17 Mean 1,8 ± 0.5 1,9 ± 0.5 1,8 ± 0.5 (*) If only day 1 or 2 is performed, this concentration is used as mean value. (LOD: Limit of Detection, Fn: False negative).

34 33 Figure 10. Laboratory results for Batch 2, 17β-nortestosterone, individual values (above) and Z-scores (below). 4,00 β-nt batch 2 (medium concentration) ppb 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0, Labcodes Mean Experiments Mean Labs +std -std crl 3 2 Z-score β -NT (medium concentration) Z-score Labcodes

35 34 Evaluation sample 2000M1844 (batch 3) for 17β-Nortestosterone. Target concentration (estimated): 4 µg 17β-Nortestosterone /l Number of participants: 16 Number of results evaluated: 16 Number of sets excluded from the evaluation 0 The individual results are summarised in Table 18. Figure 11 gives a graphical representation of the individual results and the distribution of Z-scores. Table 18. Results (µg/l) for 17β-nortestosterone: Batch 3, sample 2000M1844. Lab.no Exp. day 1 Exp. day 2 Mean(*) s.d. CV% Z-score Remarks CRL (0) 4,71 4,43 4,57 0,20 4,3 0,64 1 6,10 5,30 5,70 0,57 9,9 1,68 2 1,03 0,71 0,87 0,23 26,0-2,75 3 4,40 4,10 4,25 0,21 5,0 0,35 4 1,63 3,82 2,73 1,55 56,8-1,05 5 3,70 4,10 3,90 0,28 7,3 0,03 7 3,60 3,60 3,60 0,00 0,0-0,25 8 3,20 3,00 3,10 0,14 4,6-0,71 9 3,10 3,10 3,10 0,00 0,0-0, ,15 4,36 4,26 0,15 3,5 0, ,30 4,80 4,55 0,35 7,8 0, ,40 <LOD 4,40 0, ,25 4,55 3,40 1,63 47,8-0, ,12 4,09 4,11 0,02 0,5 0, ,50 4,00 5,25 1,77 33,7 1, ,18 4,00 3,59 0,58 16,2-0, ,20 4,60 4,40 0,28 6,4 0,49 Mean 3,8 ± 1.4 3,9 ± 1.1 3,8 ± 1.1 (LOD: Limit of Detection, Fn: False negative).

36 35 Figure 11. Laboratory results for Batch 3, 17β-Nortestosterone, individual values (above) and Z-scores (below). β-nt batch 3 (high concentration) ppb 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0, Labcodes Mean Experiments Mean Labs crl +std -std Z-score β-nt (high concentration) Z-score 2 1,5 1 0,5 0-0,5-1 -1,5-2 -2, Labcodes

37 36 Evaluation sample 2000M1845 (batch 4) for 17β-Nortestosterone. Target concentration (estimated): < 0,1 µg 17β-Nortestosterone /l Number of participants: 23 Number of results evaluated: 16 Number of sets excluded from the evaluation 0 The individual results are summarised in Table 19. Table 19 Results (µg/l) for 17ß-nortestosterone: Batch 4, sample 2000M1845. Lab.no Day 1 Day 2 Mean(*) s.d. CV% Z-score Remarks CRL (0) <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD

38 37 General summary for Nortestosterone Table 20 summarizes the average values obtained during the entire study: Results for 17α-nortestosterone (mean ± sd, µg/kg) Sample id. After lyophilisation Stability study CRL-value during p.test Overall mean during p.test 2000M1845 < 0,1 <0,1 <0,1 < 0,1 2000M1842 0,65 ± ,4 ± 0,1 0,64 ± 0,02 1,4 ± 0,6 2000M1843 3,3 ± 0.4 4,0 ± 0,3 3,3 ± 0,1 3,3 ± 1,2 2000M1844 8,1 ± ,3 ± 0,8 8,1 ± 0,1 8,2 ± 3,7 Evaluation results proficiency test 17α-nortestosterone (mean ± sd, µg/kg) Sample id. Overall Outlier False 0 Z 1 1 Z 2 Z > 2 mean Prof. test neg. 2000M1845 < 0,1 2000M1842 1,4 ± 0, M1843 3,3 ± 1, M1844 8,2 ± 3, Results for 17β-nortestosterone (mean ± sd, µg/kg) Sample id. After lyophilisation Stability study CRL-value during p.test Overall mean during p.test 2000M1845 < 0,1 < 0,1 < 0,1 < 0,1 2000M1842 0,68 ± 0,08 0,22 ± 0,08 0,65 ± 0,06 0,52 ± 0, M1843 2,1 ± 0,1 1,9 ± 0,3 2,1 ± 0,1 1,8 ± 0,5 2000M1844 4,7 ± 0,2 4,5 ± 0,5 4,6 ± 0,2 3,8 ± 1,1 Evaluation results proficiency test 17β-nortestosterone (mean ± sd, µg/kg) Sample id. Overall Outlier False 0 Z 1 1 Z 2 Z > 2 mean Prof. test 2000M1845 < 0,1 2000M1842 0,52 ± 0, M1843 1,8 ± 0, M1844 3,8 ± 1,

39 38 Evaluation sample 2000M1842 (batch 1) for 17α-Boldenone. Target concentration (estimated): 1,3 µg 17α-Boldenone /l Number of participants: 10 Number of results evaluated: 9 Number of sets excluded from the evaluation 1 The individual results are summarised in Table 21. Figure12 gives a graphical representation of the individual results and the distribution of Z-scores Table 21. Results (µg/l) Batch 1, sample 2000M1842, 17α-Boldenone Lab.no Exp. day 1 Exp. day 2 Mean(*) s.d. CV% Z-score Remarks CRL (0) 0,64 1,02 0,83 0,27 32,4-0,91 1 1,50 1,10 1,30 0,28 21,8-0,63 2 <LOD 0,28 0,28-1, ,55 6,75 9,65 4,10 42,5 Outlier 4 2,12 1,47 1,80 0,46 25,6-0,33 5 1,40 4,00 2,70 1,84 68,1 0,21 8 6,10 2,70 4,40 2,40 54,6 1,23 9 4,70 5,20 4,95 0,35 7,1 1, ,36 2,51 2,44 0,11 4,4 0, ,90 4,30 4,10 0,28 6,9 1, ,40 1,00 0,70 0,42 60,6-0,99 Mean 2,6 ± 1,9 2,4 ± 1,7 2,4 ± 1.7 (*) If only day 1 or 2 is performed, this concentration is used as mean value. (LOD: Limit of Detection, Fn: False negative).

40 39 Figure 12. Laboratory results for Batch 1, 17β-Boldenone, individual values (above) and Z-scores (below). 17α-Boldenone batch 1 (Low concentration) 8,00 7,00 6,00 5,00 ppb 4,00 3,00 2,00 1,00 0, Labcodes Mean Experiments Mean Labs crl +std -std Z-score α-boldenone (Low concentration) 2 1,5 1 Z-score 0,5 0-0,5-1 -1, Labcodes

41 40 Evaluation sample 2000M1842 (batch 1) for 17α-Boldenone. Target concentration (estimated): 4,8 µg 17α-Boldenone /l Number of participants: 10 Number of results evaluated: 8 Number of sets excluded from the evaluation 2 The individual results are summarised in Table 22. Figure 13 gives a graphical representation of the individual results and the distribution of Z-scores. Table 22. Results (µg/l) Batch 2, sample 2000M1843, 17α-Boldenone Lab.no Exp. day 1 Exp. day 2 Mean s.d. CV% Z-score Remarks CRL (0) 4,50 5,64 5,07 0,81 15,9-0,26 1 5,50 3,90 4,70 1,13 24,1-0,35 2 1,26 1,70 1,48 0,31 21,0-1, ,90 16,90 20,40 4,95 24,3 Outlier 4 6,36 5,69 6,03 0,47 7,9-0, ,30 9,90 10,60 0,99 9,3 1, ,10 10,10 11,60 2,12 18,3 1,33 9 >10 >10 > 10 Not used 11 9,46 9,24 9,35 0,16 1,7 0, ,10 10,50 10,30 0,28 2,7 1, ,35 2,50 1,93 0,81 42,2-1,03 Mean 7,0 ± 4,3 6,6 ± 3,4 6,2 ± 4,1 (*) If only day 1 or 2 is performed, this concentration is used as mean value. (LOD: Limit of Detection, Fn: False negative).

42 41 Figure 13. Laboratory results for Batch 2, 17α-Boldenone, individual values (above) and Z-scores (below). α-boldenone batch 2 (Medium concentration) 16,00 14,00 12,00 10,00 ppb 8,00 6,00 4,00 2,00 0, Labcodes Mean Experiments Mean Labs crl +std -std Z-score α-boldenone (Medium concentration) 1,5 1 0,5 Z-score 0-0,5-1 -1, Labcodes

43 42 Evaluation sample 2000M1844 (batch 3) for 17α-Boldenone. Target concentration (estimated): 12,5 µg 17α-Boldenone /l Number of participants: 10 Number of results evaluated: 8 Number of sets excluded from the evaluation 2 The individual results are summarised in Table 23. Figure 14 gives a graphical representation of the individual results and the distribution of Z-scores. Table 23. Results Batch 3, sample 2000M1844, 17α-Boldenone. Lab.no Exp. day 1 Exp. day 2 Mean s.d. CV% Z-score Remarks CRL (0) 10,06 12,42 11,24 1,67 14,8-0, ,20 17,40 20,30 4,10 20,2 0,32 2 0,37 1,12 0,75 0,53 71,2-1, ,40 39,70 48,05 11,81 24,6 Outlier 4 14,23 14,19 14,21 0,03 0,2-0, ,80 20,60 25,70 7,21 28,1 0, ,60 21,30 21,45 0,21 1,0 0,43 9 >10 >10 > 10 Not used 11 28,98 22,51 25,75 4,57 17,8 0, ,40 31,20 29,30 2,69 9,2 1, ,30 7,65 6,48 1,66 25,7-1,11 mean 18 ± ± 9 17 ± 10 (*) If only day 1 or 2 is performed, this concentration is used as mean value. (LOD: Limit of Detection, Fn: False negative).

44 43 Figure 14. Laboratory results Batch 3, 17α-Boldenone, individual values (above) and Z-scores (below). α-boldenone batch 1 (High concentration) ppb 35,00 30,00 25,00 20,00 15,00 10,00 5,00 0, Labcodes Mean Experiments Mean Labs crl +std -std Z-score α-boldenone (High concentration) Z-score 1,5 1 0,5 0-0,5-1 -1, Labcodes

45 44 Evaluation sample 2000M1845 (batch 4) for 17α-Boldenone. Target concentration (estimated): < 0,1 µg 17α-Boldenone /l Number of participants: 10 Number of results evaluated: 10 Number of sets excluded from the evaluation 0 The individual results are summarised in Table 11. Table 24. Results (µg/l) for 17α-Boldenone: Batch 4, sample 2000M1845. Lab.no Day 1 Day 2 Mean\ s.d. CV% Z-score Remarks CRL (0) <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD

46 45 Discussion results for 17α-Boldenone 17α-Boldenone is one of the analytes recently added to National Control programmes within the EU. It is demonstrated, also in this study, that it is the major metabolite of 17β-Boldenone. However, also reports on the natural occurrence have been published. Excluding the CRL in total 10 laboratories reported results. The status of these laboratories is summarised in Table 25. Table 25: Overview results in terms of category participant (N=15) EU NRL EU RFL EU-ISC Reports NON-EU NRL False negative low levels False negative higher levels No quantification 1 High levels Outlying 1 quantification Adequate results NRL: RFL: ISC: NON-EU NRL: National Reference Laboratory Routine/Field Laboratory Industry Self Control Laboratory (officially part of national Residue Control Programme) National Reference Laboratory candidate EU Member State In total seven EU NRLs reported their results. Together they represent six Member States. One additional Member State was represented through two RFLs. One NRL did only analyse the samples for the parent compound 17β-Boldenone. From a control point of view this is critical, from an analytical point of view this can adjusted relative simply. It is concluded that only a relative small number of Member States at this moment perform residue control for Boldenone. Two laboratories, including one NRL, performed analyses for the parent compound 17β-Boldenone only. In view of the current uncertainties with respect to the possible natural occurrence of 17α- and 17β- Boldenone it is suggested to give a relative low priority to the incorporation of Boldenone in National Residue Control programmes, pending a stronger scientific basis.

47 46 Evaluation sample 2000M1842 (batch 1) for 17β-Boldenone. Target concentration (estimated): 0,35 µg 17β-Boldenone /l Number of participants: 12 Number of results evaluated: 7 Number of sets excluded from the evaluation 5 The individual results are summarised in Table 25. Figure 15 gives a graphical representation of the individual results and the distribution of Z-scores. Table 25. Results Batch 1, sample 2000M1842, 17β-Boldenone. Lab.no Exp. day 1 Exp. day 2 Mean s.d. CV% Z-score Remarks CRL (0) 0,30 0,40 0,35 0,07 20,2-0,71 1 0,50 0,30 0,40 0,14 35,4-0,59 2 0,53 0,34 0,44 0,13 30,9-0,51 3 <LOD <LOD <LOD Fn 4 1,02 0,67 0,85 0,25 29,3 0,46 5 0,50 1,00 0,75 0,35 47,1 0,23 8 <LOD <LOD <LOD Fn 9 <LOD <LOD <LOD Fn 11 0,47 0,50 0,49 0,02 4,4-0,39 15 <LOD <LOD <LOD Fn 17 1,75 1,60 1,68 0,11 6,3 2,42 Outlier 20 0,51 0,14 0,33 0,26 80,5-0,77 23 <LOD 0,60 0,60-0,12 Mean 0,55±0.22 0,49±0.27 0,52±0.19 (LOD: Limit of Detection, Fn: False negative).

48 47 Figure 15. Laboratory results Batch 1, 17β-Boldenone, individual values (above) and Z-scores (below). β-boldenone batch 1 (Low concentration) 1,20 1,00 0,80 ppb 0,60 0,40 0,20 0, Labcodes Mean Experiments Mean Labs crl +std -std Z-score β-boldenone (Low concentration) 2 1,5 1 Z-score 0,5 0-0,5-1 -1, Labcodes

49 48 Evaluation sample 2000M1843 (batch 2) for 17β-Boldenone. Target concentration (estimated): 1,67 µg 17β-Boldenone /l Number of participants: 12 Number of results evaluated: 11 Number of sets excluded from the evaluation 1 The individual results are summarised in Table 26. Figure 16 gives a graphical representation of the individual results and the distribution of Z-scores Table 26. Results (µg/l) Batch 2, sample 2000M1843, 17β-Boldenone Lab.no Exp. day 1 Exp. day 2 Mean(*) s.d. CV% Z-score Remarks CRL (0) 0,78 0,94 0,86 0,11 13,2-1,48 1 1,70 1,00 1,35 0,49 36,7-0,51 2 1,73 1,90 1,82 0,12 6,6 0,40 3 <LOD <LOD <LOD Fn 4 1,75 1,44 1,60 0,22 13,7-0,03 5 1,90 2,30 2,10 0,28 13,5 0,96 8 1,40 1,20 1,30 0,14 10,9-0,61 9 2,60 2,20 2,40 0,28 11,8 1, ,98 1,99 1,99 0,01 0,4 0, ,50 0,90 0,70 0,28 40,4-1, ,65 2,25 1,95 0,42 21,8 0, ,55 1,23 1,39 0,23 16,3-0, ,90 1,90 1,90 0,00 0,0 0,57 Mean 1,6 ± 0.6 1,6 ± 0.6 1,6 ± 0.5 (LOD: Limit of Detection, Fn: False negative).

50 49 Figure 16 Laboratory results Batch 3, 17β-Boldenone, individual values (above) and Z-scores (below). Z-score β-boldenone (Medium concentration) Z-score 2 1,5 1 0,5 0-0,5-1 -1, Labcodes β-boldenone batch 1 (Medium concentration) 3,00 2,50 2,00 ppb 1,50 1,00 0,50 0, Labcodes Mean Experiments Mean Labs crl +std -std

51 50 Evaluation sample 2000M1844 (batch 3) for 17β-Boldenone. Target concentration (estimated): 4,0 µg 17β-Boldenone /l Number of participants: 12 Number of results evaluated: 12 Number of sets excluded from the evaluation 0 The individual results are summarised in Table 27. Figure 17 gives a graphical representation of the individual results and the distribution of Z-scores Table 27. Results Batch 3, sample 2000M1844, 17β-Boldenone Lab.no Exp. Day 1 Exp. day 2 Mean s.d. CV% Z-score Remarks CRL (0) 1,82 2,13 1,98 0,22 11,1-1,40 1 7,70 5,10 6,40 1,84 28,7 1,01 2 5,46 4,55 5,01 0,64 12,9 0,25 3 <LOD 4,00 4,00-0,30 4 3,30 3,04 3,17 0,18 5,8-0,75 5 5,40 5,30 5,35 0,07 1,3 0,44 8 5,50 4,20 4,85 0,92 19,0 0,17 9 8,10 9,90 9,00 1,27 14,1 2, ,51 5,52 5,52 0,01 0,1 0, ,80 4,30 4,05 0,35 8,7-0, ,20 3,10 2,65 0,64 24,0-1, ,72 3,32 3,02 0,42 14,0-0, ,90 4,20 4,05 0,21 5,2-0,27 Mean 4,5 ± 2.1 4,5 ± 1.9 4,5 ± 1.9 (*) If only day 1 or 2 is performed, this concentration is used as mean value. (LOD: Limit of Detection).

52 51 Figure 17. Results Batch 3, 17β-Boldenone, individual values (above) and Z-scores (below). β-boldenone batch 1 (High concentration) 12,00 10,00 8,00 ppb 6,00 4,00 2,00 0, Labcodes Mean Experiments Mean Labs crl +std -std Z-score β-boldenone (High concentration) Z-score 3 2,5 2 1,5 1 0,5 0-0,5-1 -1, Labcodes