1. Introduction 2. Scope 3. Field of Application 4

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INDEX Chapter:

1. INTRODUCTION 2. SCOPE 3. FIELD OF APPLICATION 4. REFERENCES 5. PRINCIPLE OF THE METHOD 6. MATERIALS 7. PREPARATION OF THE PRIMARY EXTRACT 8. EXTRACT CLEAN-UP 9. GC-MSD ANALYSIS 10. INTERPRETATION AND CALCULATION

11. VALIDATION RESULTS S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 1 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 1. INTRODUCTION Throughout the EU the use of xenobiotic anabolic agents is prohibited in food producing animals. Also the Maximum Residue Levels (MRL) for residues of these anabolics in animal products are zero (non-detectable). Analytical strategies are needed for monitoring the use by checking biological samples. 2. SCOPE This method of analyses describes the detection and conformation of the presence of anabolics in samples of animal origin. Within the field of application only semi-quantitative methods are needed. However, when deuterated internal standards are available and the purity of the standard used for identification and calibration is known, the method can be considered quantitative. 3. FIELD OF APPLICATION Table 1 shows the field of application of the described method Table 1 Field of application Analyte bovine bovine bovine limit of limit of identification muscle skin (kidney) detection (µg/kg) fat (µg/kg) Methylboldenone x x 2-5 2-5 Chloromadionone x x 2-5 2-5 acetate Chlorotestosterone x x 2 5 acetate Delmadinone x x 2 5 ß-Ethynyl estradiol x x 2 2-5 ß-Estradiol x x 2 2-5 Medroxyprogesterone x x 2 2-5 acetate Megestrol acetate x x 2 2-5 Melengestrol acetate x x 5 5 Norethandrolone x x 2 2-5 Norgestrel x x 2 2-5 ß-Nortestosterone x x 2 2-5 ß-Testosterone x 2 2-5 4. REFERENCES Herbold H.A. A multi residue method using coupled-column HPLC and GC-MS for detection of anabolic compounds in samples of urine. Standard Operating Procedure ARO/401, date 22-10-1997, RIVM. Maxwell, R.J., Lightfield, A.R., Stolker, A.A.M. (1995) An SPE column-teflon sleeve assembly for in-line retention during supercritical fluid extraction of analytes from biological matrices. J. High Resolut. Chromatogr., 18, 231-234. S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 2 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 Rossum H.J. van, Zoontjes P.W., Schwillens P.L.W.J. Multi residue analysis of anabolic agents. Standard Operating Procedure ARO/113, revision no. 5, date 20-1-1997, RIVM. S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 3 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 Stolker, A.A.M., Sipoli Marques, M.A., Zoontjes, P.W., Ginkel, van L.A., Maxwell, R.J. (1996) Supercritical fluid extraction of residues of veterinary drugs and growth-promoting agents from food and other biological matrices. Semin. Food Anal., 1(2), 101-116. Stolker, A.A.M., Zoontjes, P.W., Ginkel, van L.A. and Maxwell, R.J. (1997) Comparison of a liquid extraction and a supercritical fluid extraction for the analysis of gestagens in samples of bovine fat. In Proc. 19th Int. Symp. on CC&CE, May 1997, Wintergreen, VA, USA, 662-663. Zoontjes P.W. Analysis of kidney fat samples for gestagens. Standard Operating Procedure ARO/399, revision no. 1, date 04-10-1996, RIVM. Zoontjes P.W., Stolker A.A.M., Stephany R.W., Ginkel L.A. (1996) Onderzoek naar het gebruik van medroxyprogesteron-, chloormadinon-, megestrol- en melengestrol-acetaat in slachtdieren (in Dutch, summary in English) RIVM Intern report. 573005.004. 5. PRINCIPLE OF THE METHOD After the addition of internal (deuterated) standards, residues of anabolic agents are extracted from samples of animal tissue by supercritical fluid. After the extraction (for some of the analytes of interest) a hydrolysis is performed. The analytes are derivatised to TMS or HFB derivatives. Finally the samples are analysed by GC-MSD. Details of the sample handling depends on the matrix/analyte combination. 6. MATERIALS Reference to a company and/or product is for purposes of identification and information only and does not imply approval or recommendation of the company and/or the product by the National Institute of Public Health and Environment (RIVM) to the exclusion of others which might also be suitable. 6.1 Standards The compounds used for identification and calibration are registrated (ARO-MIS database). From these standard preparations, stock solutions containing 1 g/l (mg/ml) are prepared. These solutions are registered and stored in the dark at approximately -20°C (not higher than -10°C) for a period of 5 years. Working solutions are prepared by 10-fold dilutions of the 1 mg/ml solutions. These solutions (concentration range from 0.1 g/l to 1 ng/µl) are stored in the dark at approximately 4°C (range 1-10°C) for a maximum period of 6 months. The use of EU/CRL certified standards is recommended. Relevant standards are listed in Table 2. S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 4 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 Table 2 Standards Analyte mw Source (RIVM) Methylboldenone 286.4 Methylboldenone-d3 303.4 95M0465 Chloromadionone acetate 404.9 Chloromadionone acetate-37Cl 406.9 94M5747 Chlorotestosterone acetate 364.2 94M4509 Chlorotestosterone acetate-d3 367.2 94M0586 Delmadinone 358.1 95M1934 Ethynyl Estradiol 296.4 Ethynyl Estradiol-d4 300.4 H146344 ß-Estradiol 272.2 ß-Estradiol-d3 275.2 89M1691 Medroxyprogesterone acetate 386.3 Medroxyprogesterone acetate-d3 389.2 H148155 Megestrol acetate 384.4 Megestrol acetate-d3 387.2 94M0615 Melengestrol acetate 398.2 H153456 Melengestrol acetate-d3 401.2 94M1408 Norethandrolone 302.0 H150605 Norgestrel 312.4 H148625 Nortestosterone 274.4 Nortestosterone-d3 277.4 87M1056 ß-Testosterone 288.4 ß-Testosterone-d2 290.4 89M1692 6.2 Chemicals All listed chemicals are of Pro Analyse quality or better, unless stated otherwise. Solutions are stored at room temperature and expires 6 mounds after preparation. Water is Milli-Q water. 6.2.1 Extrelut® (Merck, 1.11738) 6.2.2 CO2 SFC/SFE grade without helium head pressure (Air Products) 6.2.3 CO2 SFC/SFE grade with helium head pressure (Air Products) 6.2.4 Methanol (Merck, 6007) 6.2.5 Acetone (Merck, 14) 6.2.6 Ethanol (Merck, 983) 6.2.7 Tert-Butylmethylether (Merck, 1845) 6.2.8 Sodium hydroxide (Merck, 6498) 6.2.9 Hydrochloric acid, 37% solution (Merck, 317) 6.2.10 Acetic acid (Merck, 63) 6.2.11 Sodium acetate (Merck, 6268) 6.2.12 Potassium hydroxide (Merck, 5033) 6.2.13 Subtilisin A (Sigma, P-5380) 6.2.14 MSTFA (N-methyl-N-trimethylsilyl-trifuor(o) acetamide from Alltech, 18061) S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 5 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 6.2.15 Heptafluorbutyric acid anhydride (HFBA), (Pierce, 63163). 6.2.16 Petroleum ether (Merck, 1775) 6.2.17 Aluminium oxide, Neutral, Activity grade 1, type WN-3 (Sigma, A-9003) 6.2.18 Tris(hydroxymethyl)-aminomethane (Merck, 8382) 6.2.19 Thiomersal (BDH, 304162) 6.2.20 Sodium chloride (BDH, 10241) 6.2.21 Polypropylene wool 6.2.22 Acetate buffer, 2 mol/l, pH 5.2. Dissolve 25.2 g acetic acid (6.2.10) and 129.5 g sodium acetate (6.2.11) in 800 ml of water. Adjust the pH at 5.2 ± 0.1 and add water to a final volume of 1000 ml. 6.2.23 Tris buffer, 0.1 mol/l, pH 9.5. Dissolve 12.1 g Tris(hydroxymethyl)-aminomethaan (6.2.18) in 800 ml of water. Adjust the pH at 9.5 ± 0.1 add water to a final volume of 1000 ml. 6.2.24 Alkaline hydrolysis solution. Dissolve 5.6 g potassium hydroxide (6.2.12) in 100 ml of methanol 6.2.25 Acidic buffer. Mix 1.7 ml hydrochloric acid (6.2.9) with 98.3 ml 2 mol/l buffer (6.2.22). 6.2.26 Methanol / water (65/35, (v/v)). Add to 65 ml of methanol (6.2.4) water to a final volume of 100 ml. 6.3 Apparatus Standard laboratory glassware and equipment is used, with in addition: 6.3.1 Glass centrifuge tubes 50 ml. 6.3.2 Automatic pipettes (Gilson P100, P200, P1000 and P5000). 6.3.3 Glass vials 2.0 ml with screw caps (Packard). 6.3.4 Refrigerated centrifuge RC3 with swing-out rotor HL-8 (Sorvall). 6.3.5 SFE extractor, Spe-ed 680 see figure 1 (Applied Separations, Applied Science Group). 6.3.6 Extraction vessels (24 ml each) and polypropylene sleeves for 3 ml SPE columns (Applied Separations, Applied Science Group) 6.3.7 Freeze-drier (Christ, Epsilon 2). 6.3.8 Centrifuge tubes, glass (55 mm x 11,5 mm) (Renes, RB55) 6.3.9 Electric water bath with nitrogen facility and thermostat adjustable ± 5oC (GFL). 6.3.10 Glass derivatisation vials (Chromacol, 2SV (A)) with screw caps (Chromacol, 8-SC) and septa (Chromacol, 8-ST15). 6.3.11 Incubator thermostat adjustable ± 5°C (Salvis). 6.3.12 Heating module with nitrogen facility and thermostat adjustable ± 5°C (Pierce, 18790). 6.3.13 Glass inject vials (Chrompack, art. no. 10201) with glass inserts (Chrompack, 10381). 6.3.14 GC-MSD system (Hewlett-Packard). Model 5890 Series II Plus Gas Chromatograph, Model 7673 auto-injector and a Model 5972 mass-selective detector. The steroids were analysed on a CP-SIL 5CB column, 25 m x 0.25 mm ID, film thickness 0.12µm (Chrompack). 6.3.15 Centrifuge tubes (50 ml), glass (Pyrex, Z14,588-2) 6.3.16 pH-meter ASA2000 (Applikon) 6.3.17 Vortex (Vortex-genie) 6.3.18 Baker-21 SPE-extraction system (Baker) S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 6 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 7. PREPARATION OF THE PRIMARY EXTRACT The preparation of the primary (SFE)-extract can be divided in three steps: - pre-SFE, - SFE, - post-SFE. 7.1 Pre-SFE Before performing SFE some sample handling is necessary 7.1.1 Muscle: Freeze-dry 5 grams of tissue sample conform the general freeze-drying procedure (conform SOP ARO/404). Blend the freeze-dried sample with 3.5 gram Extrelut (6.2.1) in a beaker glass (50 ml). Add the internal standard solutions (25 µl of standard solution 1.0 ng/µl) and add 1 ml of water. 7.1.2 Skin: From the laboratory sample a test portion of 5.0 g is weighted into a 50 ml glass centrifuge tube (6.3.1). Add the internal standard solutions (25 µl of standard solution 1.0 ng/µl). After at least 30 min, add 20 ml 0.1 mol/l Tris-buffer, pH 9.5, containing 5 mg Subtilisin A (6.2.13). The enzymatic digestion is performed to free the analytes from cells and from conjugates with proteins (non covalently protein bound residues). After the enzymatic digestion the sample is cooled to room temperature and extracted twice with 20 ml of TBME (6.2.7). The TBME is evaporated. The dry residue is redissolved in 3 ml of TBME. Pour the TBME extract on 3.5 gram of Extrelut (6.2.1) sorbent in a beaker glass (50 ml). Wash the solvent tube with a second portion of 3 ml of TBME and pour the TBME on the Extrelut mixture. Wait for 30 min until the TBME is evaporated. Add 1 ml of water. 7.1.3 Fat: Homogenise the fat sample by cutting the sample in small cubes. Blend 2 grams of sample with 3.5 grams of Extrelut (6.2.1) sorbent; add the internal standard solutions (10 µl of standard solution 1 ng/µl) and 1 ml of water. 7.2 SFE-Extraction Legend for Figure 1: Schematic of the in-line trapping technique, a) SPE-column, b) Column sleeve,

c) Extraction vessel configured for in-line trapping. SF-CO2 enters the bottom of the extraction vessel, diffuses through the sample matrix and solubilises target analyte(s) and endogenous co-extractables, and then contacts the in-line sorbent bed, where the analytes are adsorbed from the SF and retained. S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 7 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30

Figure 1 Aluminium oxide SPE column 7.2.1 Packing the SFE vessel. 7.2.1.1 Attach one end-cap fitting to the Keystone extraction vessel and seal. This end-cap is labelled TOP. 7.2.1.2 Fill the filtration column (a in Figure 1) with 2 grams of neutral aluminium oxide (6.2.17) and fill the empty space of the column with a plug of polypropylene wool (6.2.21). Trim the finger grip flange of the column slightly so that it fit within the walls of the extraction vessel. 7.2.1.3 Insert the SPE column into the Teflon sleeve (b in Figure 1) until the Luer tip protrudes from the sleeve bottom. 7.2.1.4 Insert the whole assembly Figure 1 (a+b) into the extraction vessel (c in Figure 1) and force the assembly to the capped end of the vessel with a tamping rod. Insert a plug of polypropylene wool (approximately 1 cm think) into the column and tamp firmly. 7.2.1.5 Pour the sample mixture into the column and tamp firmly. Fill the remaining void in the column with a second plug of polypropylene wool and tamp firmly. 7.2.1.6 Screw the end-cap (labelled BOTTOM) in place and seal. 7.2.1.7 Install the extraction column vertically in the SFE oven with the end labelled TOP connected to the outlet shut-off valve (see Figure 2). S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 8 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 7.2.2 Extraction of anabolic agents

Figure 2: Diagram of the Spe-ed 680 Supercritical Fluid Extractor a) inlet valve b) micro-metering valves c) outlet valves d) vent valves S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 9 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 7.2.2.1 Close the inlet (a), outlet (b) and vent (d) valves. 7.2.2.2 Open the valve of the CO2 cylinder and switch the fluid delivery valve at the front of the Spe-ed SFE to open to start the pressurisation of the system. 7.2.2.3 Turn de big black button at the front of the pump module clockwise to increase the pressure to 650 bar. 7.2.2.4 Vent the pump by opening the vent valves (a) carefully and let the CO2-fluid flow for 15 seconds (until you see the white stream of CO2 liquid). Close the vent valves. 7.2.2.5 Heat the SFE micro-metering valve module to 110°C. 7.2.2.6 Snap the filled extractor vessel(s) into the mounting brackets. Connect the inlet and outlet fittings to the extractor vessels. 7.2.2.7 Open the two inlet valves of the oven module. Check the pressurised system for leaks before proceeding any further. 7.2.2.8 Close the oven. Begin heating the oven by switching the oven module on (heat to 50°C). 7.2.2.9 Begin to pressurise the system to the desired pressure by slowly turning the knob labelled “air pressure regulator” clockwise to increase the pressure. Continue turning the air pressure regulator knob until the system pressure on the digital display is 650 bar. 7.2.2.10 Let the system equilibrate for 10 min (static extraction period). 7.2.2.11 Make sure that the micro-metering valves (c) are nearly closed (but not completely closed). Slowly open the outlet valves (b) to direct the flow fluid to the micro-metering module. Continue the flow of CO2 until a flow of 2 L/min. Keep the flowrate on 2 L/min for 10 min (dynamic extraction period). CAUTION: The micro-metering valves are not shut off valves. Do not overtighten them when reducing flow. 7.2.2.12 After the extraction, shut off the flow of fluid by closing the inlet and the outlet valves. Open the vent valves (d) carefully and depressurises the extractor vessels. 7.2.2.13 Switch the oventemperature off. Turn the air pressure regulator knob counter-clockwise to shut off the air pressure to the pump module. The air pressure gauge will read 0 bar. After the system has been depressurised, close the vent valves. 7.2.2.14 Remove the extractor vessels from the oven. 7.2.2.15 Flush the transfer lines of the system (open the outlet valves and the micro-metering valves) by pushing 4 ml of methanol with a syringe through the tubing and valves. Table 3 SFE conditions Static extraction Dynamic extraction time (min) 10 10 oven temperature in °C 50 50 Micro-metering 110 110 temperature in °C pressure 650 450 flow (l/min) 0 2 7.3 Post-SFE 7.3.1 Remove the end-cap fittings from the extractor vessels. 7.3.2 Retrieve the in-line SPE column from the vessel by removing the spent sample matrix and pushing out the SPE-column-Teflon sleeve assembly with a tamping rod. 7.3.3 Remove the SPE-column out of the sleeve, and place the SPE- column in the SPE- extraction system (6.319). 7.3.4 Elute the SPE-column with 6x1ml of methanol/water solution (6.2.26). S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 10 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 8. HYDROLYSIS/CONCENTRATION/DERIVATISATION OF THE PRIMARY EXTRACT Schematic overview of sample handling is shown in Figure 3

Sample pre-treatment

SKIN MUSCLE perform an enzymatic FAT freeze-dry the tissue digestion followed by TBME-extraction

SFE Perform the SFE using unmodified CO2 and trap the analytes in-line on an alumina SPE-column

Elute the alumina SPE-column

MUSCLE and SKIN divide the eluate in two equal FAT portions

Alkaline hydrolysis + TBME - extraction TBME-extraction

Derivatise with MSTFA++ Derivatise with HFBA

GC-MSD analysis

Figure 3 Flow diagram of the method of analysis S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 11 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 8.1 Muscle/Skin 8.1.1 Divide the extract (from 7.3.4) in two equal portions (Portion 1 and Portion 2). 8.1.2 Portion 1: an alkaline hydrolysis (see paragraph 8.3) and a HFBA derivatisation (see paragraph 8.4) are performed. 8.1.3 Portion 2: evaporate the methanol/water solvent to dryness under a stream of nitrogen in a waterbath (6.3.9) of 50°C. Add 2 ml of water and vortex (6.3.17) for 30 sec. Add 6 ml of TBME (6.2.7). Vortex for an other 30 sec. Centrifuge (6.3.4) for 5 minutes at 2500 rpm at 20°C, and transfer the TBME to a clean tube. Repeat the procedure of TBME extraction. Combine the TBME supernatants. Evaporate the TBME to dryness under a stream of nitrogen in a waterbath of 40°C. Perform the MSTFA++ derivatisation as described in paragraph 8.5. 8.2 Primary extract of the sample of fat Perform the hydrolysis and the HFBA derivatisation as described in respectively paragraph 8.3 and 8.4. 8.3 Alkaline hydrolysis 8.3.1 Preparation of control standards: Pipette directly into empty tubes aliquots of the standard and internal standard solutions. The following amounts of standards are used: 5, 10, 20 and 30 ng of analytes respectively. The amount of the internal standard must be identical to the amount added to the samples. 8.3.2 Dry the standards under a cold stream of nitrogen in a water bath at 50°C. 8.3.3 Evaporate the solvents from the samples solutions under a cold stream of nitrogen in a water bath at 50°C. 8.3.4 The dry standards and sample residues are dissolved in 0.2 ml alkaline hydrolysis solution (6.2.24). This mixture is incubated at 37°C for 30 minutes. The hydrolysis is ended by the addition of 0.8 ml acidic buffer (6.2.25). 8.3.5 Add 6 ml TBME to the mixture. Mix by placing the tube on a Vortex for 30 seconds. Centrifuge for 5 minutes at 2500 rpm at 20°C, and transfer the TBME to a clean tube. Repeat the procedure of TBME extraction. 8.3.6 The combined supernatants (TBME) are evaporated to dryness under a stream of nitrogen in a waterbath at 40°C. 8.3.7 Perform a HFBA derivatisation (see paragraph 8.4). 8.4 HFBA-derivatisation Dissolve the dry residue in 0.4 ml ethanol by placing the tube in an ultrasonic waterbath (6.3.7) during 1 minute followed by placing on a Vortex during 130 seconds. Transfer the residue to a derivatisation vial. The ethanol is evaporated in a heating module under nitrogen. Add the 0.050 ml HFBA reaction mixture and place the tube on a Vortex during 30 seconds followed by incubation of the reaction mixture during 1 hour at 60°C. After incubation, the reaction mixture is evaporated to dryness under a stream of nitrogen at 50°C and the derivatised residue is dissolved in 25µl iso-octane by placing in an ultrasonic waterbath during 1 minute, followed by using a Vortex during 30 seconds. Transfer the iso- octane to a GC-injection-vial with micro insert. S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 12 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 8.5 MSTFA++ derivatisation Preparation of control standards: The following amounts of standards are used; 20, 30, 40 and 50 ng of analytes respectively. The amount of the internal standard must be identical to the amount added to the samples. Evaporate the solvent. Dissolve the dry residues of the samples from 8.1 and the control standards in 0.4 ml ethanol by placing the tubes in an ultrasonic waterbath during 1 minute followed by placing on a Vortex during 130 seconds. Transfer the residues to a derivatisation vial. The ethanol is evaporated in a heater module under nitrogen. The residue is derivatised with 25 µl of a mixture N-methyl-N-trimethylsilyltrifluoro-acetamide (MSTFA)-ammonium iodide- dithioerythritol (1000:2:4, v/w/w). The derivatisation-vials are placed in an oven at 60°C for 1 hour. Evaporate the derivatisation reagents at 50°C under a stream of nitrogen until dry, and dissolved the residue in iso-octane (25 µl) and transferred into a GC-injection-vial with micro insert. 9. GAS CHROMATOGRAPHY-MASS SPECTROMETRY Table 4 GC-MSD conditions HFBA-derivatives MSTFA++-derivatives column CP-SIL 5CB CP-SIL 5CB injection 5 µl splitless 5 µl splitless injector temperature 250°C 250°C initial oven temperature 75°C (1 minute) 75°C (1 minute) temperature programme 30°C/min 30°C/min final temp 210°C (0.5 min) 250°C (1.17 min) temperature transfer line 280°C 280°C solvent delay 8.35 min 8.5 min dwelltime per ion 40 msec 40 msec monitored ions: m/z 479 Medroxy- m/z 418 Nortestosterone progesterone m/z 482 Medroxy- m/z 421 Nortestosterone-d3 progesterone-d3 m/z 477 Megesterol m/z 444 Methylboldenone m/z 480 Megesterol-d3 m/z 447 Methylboldenone-d3 m/z 447 Melengestrol m/z 446 Norethandrolone m/z 450 Melengestrol-d3 m/z 456 Norgestrel m/z 497 Chloromadinone m/z 436 Chlorotestosterone acetate m/z 499 Chloromadinone-37Cl m/z 439 Chlorotestosterone acetate-d3 m/z 495 Delmadinone m/z 664 b-Estradiol m/z 667 b-Estradiol-d3 m/z 474 Ethynyl estradiol m/z 478 Ethynyl estradiol-d4 m/z 320 Testosterone m/z 322 Testosterone-d2 S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 13 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 The samples and standard solutions were analysed by GC-MS using the conditions shown in Table 4. Standards were derivatised corresponding to a mass-concentration range from 0- 10 µg/kg. In case of a positive screening result the derivatised extract was reïnjected and analysed for the presence of more ions diagnostic for the compound detected. 10 INTERPRETATION AND CALCULATION 10.1 Interpretation and calculation The first step in interpreting the results is to check: - For performance characteristics of the GC-MS system (MS-tuning), - Sensitivity for external derivatised standards, - Signals for internal standards. 10.2 Calculation of quantitative results Quantitative results are obtained by constructing calibrationcurves of the response variable versus the concentration. Quantification is only valid if`: - The maximum of the signal originating from the analyte exceeds the noise + 3 SD - The coefficient of correlation is better than 0.97 - The numerical value of the intercept does not deviate more than ± 3 SD from zero. Calibrationcurves are calculated using least squares linear regression analysis. 10.3 Calculation in case of deuterated internal standards For an increasing amount of compounds (see Table 2) deuterated internal standard are available and therefore quantification is straightforward. The area of the selected ion of the standard and internal standard are calculated and the ratio is the response variable. A calibrationcurve is constructed by analysing different concentrations of standard. A linear curve is fitted using least squares linear regression calculation. Unknown concentrations are calculated by interpolation. 10.4 Calculation without corresponding deuterated internal standard For some compounds no deuterated analogues were not validated extensively yet or not available. For these analytes (alternative) internal standards are used to control for false negative results at the limit of detection. Quantification of the analyte content results in semi quantitative (indicative) results only. 10.5 Identification. For identification according to the EC-criteria it is mandatory that at least 4 ions be monitored. Each ion monitored (response) should fulfil the criterion that the maximum exceeds the average noise + 3 SD. If this criterion is fulfilled the 3 different ratios are calculated. The same ratios are calculated for the standard analyte, preferably at the corresponding concentration. For positive identification the responses obtained for the unknown sample should preferably all be within ± 10% of the average value of the standard. S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 14 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30 Notice: Frequently the variability of the MS-equipment does not allow this criterion to be strictly applied. In this case the criterion is replaced by a criterion based on the experimental variability. The response ratios for the unknown sample should be within the corresponding standard value ± 3 sd. 10.6 Analytical follow-up If the experiment fulfils the quality control criteria: - Validated GC-MS detection, - Good recovery of internal or external standards, - No contamination in blank control samples, and the results of the analysis is negative, the experiment is closed and administrated as such. If one or more of the samples shows a response possibly related to any of the analytes the sample is reanalysed under strict conditions. For confirmation it might be necessary to repeat the analysis without the addition of an isotope enriched internal standard since fragment-ions of the internal standard can interfere with the fragment-ions of the analyte. 11. METHOD VALIDATION RESULTS The limit of detection was expressed as the concentrations at which the maximum of the signal (of the most intensive ion) originating from the analyte showed a response S/R =3. The limit of identification was defined as the concentration at which four of the most intensive ions originating from the analyte show responses S/R>=3. Repeatability and reproducibility experiments were performed by analysing fortified samples three days in triple. From the samples analysed the peak areas of analyte and internal standard were measured and the ratio analyte/IS was calculated. The mean ratio of analyte/IS for every analyte/matrix combination was calculated within the day and between the days. The repeatability was expressed as %RSD of the mean ratio’s within the days and the (within-laboratory) reproducibility was expressed as the %RSD of the mean ratio’s between the days. Table 2 shows the different analyte/matrix combinations and their limits of detection and identification. Table 5,6 and 7 give the validation results of the method. Until now not all the different combinations are validated. Table 5 Validation results of SFE/GC-MSD of gestagens from fortified bovine kidney fat analyteb repeatability in %RSD within-laboratory reproducibility (n=9) in %RSD (n=3) Medroxyprogesterone 13 39 acetate Melengestrol acetate 14 13 Chloromadinone acetate 5 7 Megestrol acetate 8 20 Delmadinone acetate 11 84a a no deuterated internal standard was available so only qualitative analysis is possible b fortification level 5 µg/kg S.O.P. : ARO/435 Title: Method of analysis for anabolic steroids Page : 15 of 14 in animal tissues using SFE-GC-MSD Revision : 0 Date : 2000.08.30

Table 6 Validation results of SFE/GC-MSD of steroids from fortified bovine muscle analyteb repeatability in within-laboratory reproducibility %RSD (n=9) in %RSD (n=3) Methylboldenone 12 23 ß-Nortestosterone 4 2 Ethynyl estradiol 7 9 Chlorotestosterone 5 (n=6) 4 acetate Norethandrolone 21 37a Norgestrel 15 31a a no deuterated internal standard was available so only qualitative analysis is possible b fortification level 5 µg/kg

Table 7 Validation results of SFE/GC-MSD of steroids from fortified bovine skin analyteb repeatability in within-laboratory reproducibility %RSD (n=9) in %RSD (n=3) ß-Estradiol 40 27 Ethynyl estradiol 11 23 (n=2) ß-Testosterone 12 (n=6) 2 (n=2) ß-Nortestosterone 6 14 Methylboldenone 42 (n=6) 32 Chlorotestosterone 10 18 acetate b fortification level 5 µg/kg