Muscle, Poultry Liver, Water and Fish

Home , Chlormadinone, Isoflupredone, Melengestrol, Trenbolone

TITLE : MUSCLE, POULTRY LIVE R, WATER AND FISH - THE SCREENING, QUANTIFICATION AND CONFIRMATION OF A SELECTION OF GROWTH PROMOTORS - LC-MS/MS

1 OBJECTIVE AND SCOPE This SOP describes the analysis using LC-MS/MS of a selection of growth-promoting compounds in muscle, poultry liver, water and fish. These compounds are: 17 α–trenbolone, 17β–trenbolone, 17 α-nortestosterone, 17 β- nortestosterone, androsta-1,4-diene-3,17-dione (ADD), 17 α–boldenone, 17 β-boldenone, methylboldenone, 17 α- testosterone, 17 β-testosterone, 17 α-methyltestosterone, 16 β-OH-stanozolol, stanozolol, prednisolone, methylprednisolone, isoflupredone, flumethasone, dexamethasone, betamethasone, triamcinolon-acetonide, clobetasol, megestrol-acetate, melengestrol-acetate, medroxyprogesterone-acetate, chlormadinone-acetate, α- zeranol ( α-zearalanol), β-zeranol ( β-zearalanol), zearalanon, zearalenon, α-zearalenol, β-zearalenol. Bovine muscle is fully validated, other muscle, poultry liver, water and fish are partially validated. The method has a measuring range of 0.2 to 5.0 µg/kg.

2 DEFINITION MMS Matrix matched standards ES Electrospray MS Mass spectrometer UPLC Ultra Performance Liquid Chromatography ADD Androsta-1,4-diene-3,17-dione SPE Solid Phase Extraction ACN Acetonitrile MeOH Methanol FA Formic acid (HCOOH) Water MilliQ H2O DMSO Dimethyl sulfoxide HAc Acetic acid NH3 Ammonia

3 PRINCIPLE The method consists of the following steps: • Destruction of meat with a bead ruptor • Extraction of analytes from destructed meat • Sample clean-up with 96-wells SPE • Analysis with LC-MSMS

4 CHEMICALS AND REAGENTS All reagents and chemicals must be at least pro analysis quality. With ‘water’ is meant water, purified with a MilliQ® system with a minimum resistance of at least 18.2 M Ω.cm -1. Instead of listed below, chemical products from other suppliers may be used, as long as they are from comparable quality.

4.1 Standards

4.1.1 17 α–Trenbolone (NMI D708) 4.1.2 17 β–Trenbolone (Steraloids E3170-000) 4.1.3 17 β–Boldenone (Steraloids A200) 4.1.4 17 α–Boldenone (Rikilt, EU/CRL 53, this is an ampoule containing 0.1 mg 17 α–boldenon) 4.1.5 17 α-Testosterone (Steraloids, A6900) 4.1.6 17 β-Testosterone-d3 (NMI, D546) 4.1.7 Prednisolone (Sigma-Aldrich P6004) 4.1.8 Methylprednisolone (Sigma-Aldrich M0639) 4.1.9 Megestrol-acetate (Sigma-Aldrich M0513) 4.1.10 Melengestrol-acetate (Sigma-Aldrich 33998) 4.1.11 Chlormadinone-acetate (Sigma-Aldrich C5145) 4.1.12 Isoflupredone (Steraloids P0553-000) 4.1.13 Flumethasone (Sigma F9507) 4.1.14 Triamcinolone-acetonide (Sigma T6501) 4.1.15 Betamethasone (Steraloids P0520-000) 4.1.16 Dexamethasone (Sigma D1756) 4.1.17 Clobetasol (Steraloids P0086-000) 4.1.18 ADD (Androsta-1,4-diene-3,17-dione) (Steraloids A100) 4.1.19 α-Zeranol (Sigma Z0292) 4.1.20 β-Zeranol (Sigma Z0417) 4.1.21 Zearalanone (TRC Z270450 this is a vial containing 1 mg zearalanon) / Sigma Z0167 4.1.22 Zearalenone (Sigma Z2125) 4.1.23 α-Zearalenol (Sigma Z0166) 4.1.24 β-Zearalenol (Sigma Z2000))

4.1.25 16 β-OH-Stanozolol (NMI D621, this is an ampul containing 1 mg 16 β-OH-stanozolol) 4.1.26 Isoflupredone-d3 (d2 major) (TRC I816602, this is an vial containing 1 mg Isoflupredone-d3 (d2 major).

4.1.27 17 β–Trenbolone-d3 (EURL 0102) (This is an ampoule containing 0.1 mg 17 β–trenbolone-d3) 4.1.28 17 β–Boldenone-d3 (EURL 0065) (This is an ampoule containing 0.1 mg 17 β–boldenone-d3) 4.1.29 α-Zeranol-d4/ β-zeranol-d4 (EURL 0006) (This is an ampoule containing 0.05 mg α-zeranol-d4 and 0.05 mg β-zeranol-d4) 4.1.30 α-Zearalenol-d4 (EURL 0048) (This is an ampoule containing 0,1 mg α-zearalenol-d4)

4.1.54 α-Zearalenol-d7 (EURL 0091) (This is an ampoule containing 0.05 mg α-zearalenol-d7) 4.1.31 β-Zearalenol-d4 (EURL 0049) (This is an ampoule containing 0,1 mg β-zearalenol-d4) 4.1.32 β-Zearalenol-d7 (EURL 0092) (This is an ampoule containing 0.05 mg β-zearalenol-d7) 4.1.33 16 β-OH-stanozolol-d3 (EURL 0064) (This is an ampoule containing 0.1 mg 16 β-OH-stanozolol-d3) 4.1.34 Triamcinolone-acetonide-d6 (EURL 0063) (This is an ampoule containing 0.1 mg triamcinolon-acetonide- d6) 4.1.35 Dexamethasone-d4 (Cachesyn, CSTD361) 4.1.36 Zearalenone-d6 (TRC Z270502) (This is an vial containing 1 mg zearalenone-d6) 4.1.37 Zearalanone-d6 (TRC 270442) (This is an vial containing 0.25 mg zearalanone-d6) 4.1.38 α-Zearalanol-d4/ β-zearalanol-d4 (EURL 0006) (This is an ampoule containing 0.05 mg α-zearalanol-d4 and 0.05 mg / β-zearalanol-d4) 4.1.39 17 β–Nortestosterone-d3 (EURL 0004) (This is an ampoule containing 0.1 mg nortestosterone-d3) 4.1.40 Melengestrol-acetate-d3 (TRC M215352) 4.1.41 Megestrol-acetate-d3 (EURL 0013) (This is an ampoule containing 0.1 mg megestrol-acetate-d3) 4.1.42 Prednisolone-d8 (major compound prednisolone-d7) (TRC P703742) 4.1.43 Medroxyprogesterone-acetate (Steraloids Q3021-000) 4.1.44 Medroxyprogesterone-acetate-d3 (EURL 011) (This is an ampoule containing 0.1 mg medroxyprogesterone-acetate-d3 4.1.45 Stanozolol (Cerilliant S-906) 4.1.46 Stanozolol-d3 (Cerilliant S-910) 4.1.47 Methylboldenone (Steraloids A0130-040) 4.1.48 Methylboldenone-d3 (EURL 52) (This is an ampoule containing 0.1 mg methylboldenone-d3) 4.1.49 17 β-Testosterone (Steraloids A6950-000) 4.1.50 17 α–Nortestosterone (Steraloids E4040-000) 4.1.51 17 β–Nortestosterone (Steraloids E4050-000) 4.1.52 17 α–Methyltestosterone (Steraloids A6280-000) 4.1.53 17 α–Methyltestosterone-d3 (EURL 05) (This is an ampoule containing 0.1 mg methyltestosterone-d3)

4.2 Chemicals 4.2.1 Ammonia 25% (Merck, 105432)

4.2.2 Acetic acid glacial 100% (Merck, 100063)

4.2.3 Formic acid 100% ULC grade (ActuAll, 806012817)

4.2.4 Methanol ULC grade (ActuAll, 813013802)

4.2.5 Acetonitrile ULC grade (ActuAll, 801022802)

4.2.6 Ethanol (100%) (Merck, 100983)

4.2.7 Water ULC grade (ActuAll, 823013802)

4.2.8 Ammonium formate (Sigma 156264)

4.2.9 Dimethylsulfoxide (DMSO) (Sigma-Aldrich D5879)

4.3 Standard solutions 4.3.1 Stock solutions 100 or 1000 mg/l Prepare individual stock solutions of each reference compound (see table below). Weigh reference compounds to 0,01 mg accurate in amber coloured vials with screw cap. Use an analytical balance. Add an amount of MeOH to obtain the concentration of 1000 mg/l. Take account of the test portion, purity and appearance of the standard substance. The amount of MeOH is added by weighing and converting the weighed amount to volume by using the density of ethanol at room temperature 0.79 g/cm ³. Internal standards expire after 5 years. Store in freezer.

name stability (weeks) Concentration (mg/l) 17 α-testosterone 104 1000 17 β-testosterone 104 1000 17 α-trenbolone 4.5 1000 17 β-trenbolone 52 1000 17 α-methyltestosterone 104 1000 17 α-nortestosterone 104 1000 17 β nortestosterone 104 1000 17 α-boldenone 4.5 100 17 β-boldenone 52 1000 methylboldenone 4.5 1000 androstadienedione 52 1000 16 β-hydroxystanozolol 52 100 stanozolol 104 1000

α-zearalanol 104 1000 β-zearalanol 104 1000 α-zearalenol 13 1000 β-zearalenol 13 1000 zearalanone 70 1000 zearalenone 104 1000

chlormadinone acetate 52 1000 medroxyprogestrone-17-acetate 52 1000 megestrol acetate 52 1000 melengestrol acetate 52 1000

betamethasone 52 100 clobetasol 156 1000 dexamethasone 156 1000 flumethasone 156 1000 isoflupredone 156 1000 methylprednisolone 156 1000 prednisolone 156 1000 Triamcinolon-acetonide 156 1000

Internal standards 260 1000

4.3.2 Mix standard solution I 1 mg/l (MSS I) Pipette of each stock solution 10 µl (1000 mg/l) or 100 µl (100 mg/l) in an amber coloured vial with screw cap. Add an amount of MeOH till an exact total volume of 10 ml final volume. The amount of MeOH is added by weighing and converting the weighed amount to volume by using the density of MeOH at room temperature 0.79 g/cm³. Store in freezer.

4.3.3 Internal standard solution 1 mg/l (ISS I) Pipette of each internal stock solution 10 µl in an amber coloured vial with screw cap. Add an amount of MeOH till an exact total volume of 10 ml final volume. The amount of MeOH is added by weighing and converting the weighed amount to volume by using the density of MeOH at room temperature 0,79 g/cm ³. Expiration 3 years. Store in freezer.

4.3.4 MSS II 100 µg/l Pipette 500 µl of MSS I (4.3.2) in an amber coloured vial with screw cap. Add 4500 µl of MeOH. Expiration 1 month. Store in freezer.

4.3.5 ISS II 100 µg/l Pipette 500 µl of ISS I (4.3.3) in an amber coloured vial with screw cap. Add 4500 µl of MeOH. Expiration 1 month. Store in freezer.

4.3.6 MSS III 10 µg/l Pipette 500 µl of MSS II (4.3.4) in an amber coloured vial with screw cap. Add 4500 µl of MeOH. Expiration 1 month. Store in freezer.

4.4 Reagents 4.4.1 Ammonium formate 1M Dissolve 6.3 g ammonium formate in water and add water to a final volume of 100 ml. This solution is stable for 3 months at room temperature.

4.4.2 Wash solvent 1: 60% MeOH / 2% HAc (500 ml) Mix 190 ml water, 300 ml MeOH and 10 ml HAc. This solution is stable for 6 months at room temperature.

4.4.3 Wash solvent 2: 20% ACN / 2% HAc (500 ml) Mix 390 ml water, 100 ml ACN and 10 ml HAc. This solution is stable for 6 months at room temperature.

4.4.4 Wash solvent 3: 20% ACN (500 ml) Mix 400 ml water with 100 ml ACN. This solution is stable for 6 months at room temperature. 4.4.5 Wash solvent 4: 10% ACN / 2% NH3 (500 ml) Mix 410 ml water, 40 ml ammonia (25%) and 50 ml ACN. This solution is stable for 6 months at room temperature.

4.4.6 Wash solvent 5: 50% MeOH / 2% NH3 (500 ml) Mix 210 ml water, 40 ml ammonia (25%) and 250 ml MeOH. This solution is stable for 6 months at room temperature.

4.4.7 Wash solvent 6: 60% MeOH (500 ml) Mix 200 ml water and 300 ml MeOH. This solution is stable for 3 months at room temperature.

4.4.8 Mobile phase A: water / ACN / FA / ammonium formate 1M 900 / 100 / 0.02 / 2 (v/v/v/v) Mix 900 ml water, 100 ml ACN, 0.02 ml FA and 2 ml ammonium formate 1 M. This solution is stable for 1 month at room temperature.

4.4.9 Mobile phase B: water / ACN / FA/ ammonium formate 1M 100 / 900 / 0.02 / 2 (v/v/v/v) Mix 100 ml water, 900 ml ACN, 0.02 ml FA and 2 ml ammonium formate 1 M. This solution is stable for 6 months at room temperature.

4.4.10 Extraction solvent: 60% ACN (500 ml) Mix 200 ml water and 300 ml ACN. This solution is stable for 3 months at room temperature.

5 EQUIPMENT Any reference to type and/or product is only to inform the user and identify the equipment and does not imply exclusion of similar equipment.

5.1 Analytical balance with an accuracy of 0.01 mg or better (Mettler/Toledo AX205)

5.2 Vortex (IKA Genius 3)

5.3 Bead Ruptor (Omni International, Bead Ruptor 24) with 7 ml tube carriage kit (19-010-320)

5.4 Empty sample tubes 7 ml (LA Biosystems, 19-651)

5.5 Beads 2.3 mm zirconia/silica (BioSpec Products 11079125z)

5.6 Centrifuge (Heraeus Multifuge 3L) with large reaction tube module (5 tubes per module)

5.7 Clean glass test tube of 14 ml, 100 x 15 x 0.85 (Beldico 8739007)

5.8 Extraction plate manifold for Oasis 96-wells plates (Waters 186001831)

5.9 Vacuum pump (KNF Lab Laboport D79112)

5.10 Oasis HLB 96-well plate, 60 mg sorbent per well, 60 µm particle size (Waters 186000679)

5.11 Evaporator for 96 wells (Biotage TurboVap 96)

5.12 96-Well collection plates for Waters Acquity UPLC I-Class, well volume 2 ml (Waters 186002482)

5.13 96-Well PTFE/Silicone seal with pre-slit (Waters 186006335)

5.14 12-Channel pipette (Rainin 100-1200 µl LTS Pipet Lite, L12-1200XLS,17013810)

5.15 12-Channel pipette (Rainin 20-200 µl LTS Pipet Lite, L12-200XLS, 17011835)

5.16 UPLC-MS/MS system containing: 5.16.1 Waters ACQUITY UPLC® I-Class System

5.16.2 Analytical column: Acquity UPLC BEH C 18 , 1.7 µm, 1.0 x 100mm (Waters 186002346) 5.16.3 Waters triple quadrupole mass spectrometer Xevo TQ-S with ESI interface

PROCEDURE 6.1 General This SOP describes the quantification and confirmation of a selection of growth promotors in bovine consumption meat. Sample clean-up is performed using a bead ruptor machine and 96-wells SPE. In conventional methods, digestion of meat is performed enzymatically or mechanically (SOP A1160, ultrasonification) or shaking thoroughly with acetonitrile (SOP A1172). In this SOP another mechanically digestion is implemented. Digestion/disrupting, homogenisation and liquid extraction is carried out by means of a bead ruptor machine. This bead ruptor is specifically designed for grinding,

lysing, and homogenizing biological samples prior to molecular extraction. In ‘bead beating’, a large number of minute glass, ceramic or steel beads are vigorously agitated by shaking or stirring. Disruption occurs by the crushing action of the beads as they collide with the cells. The method has been used for years to disrupt microorganisms. More recently, bead mill homogenization has been applied to soil samples and to small samples of plant and animal tissue. The size of the beads used is important. Optimal size for bacteria and spores is 0.1 mm , 0.5 mm for yeast, mycelia, microalgae, and unicellular animal cells such as leucocytes or trypsinized tissue culture cells and 1.0 or 2.5 mm for tissues such as brain, muscle, leaves and skin. Speed of disruption is increased about fifty percent by using like-sized, heavier ceramic beads made of zirconia-silica or zirconia rather than glass. Really tough tissue sometimes requires chrome-steel beads - which are 5 times more dense than glass beads. After treatment, the beads settle by gravity in seconds and the cell extract is easily removed and further processed. In this method the tubes used are centrifuged, the supernatant is transferred to the conditioned 96-well SPE plate. The SPE plate is washed with different solvents after which the compounds of interest are eluted and transferred to the LC-MS/MS for further analysis.

6.2 Safety precautions Take precautions to prevent inhalation and contact with skin from standards and chemicals. Work in a fume hood and wear gloves if necessary (SOP F0071).

6.3 Sample pre-treatment 6.3.1 Preparing samples - Samples are stored in the freezer after grinding and homogenizing. Before analysis, the samples are slowly thawed to room temperature. An aliquot of 500 mg (± 50 mg) is transferred to a 7 ml tube (5.4). An aliquot of 500 µl (± 50 µl) water sample is transferred to a clean glass test tube (5.7).

6.3.2 Preparing sample tubes - Add a single layer of beads to the 7 ml tubes (5.4).

6.4 Amount of sample 500 mg or 500 µl (water) is processed. Don’t use tap water as a blanc sample.

6.5 Sample clean-up 6.5.1 Primary extraction - Weigh 1 test unit (0.5 g) of every homogenized sample in 7 ml polypropylene tubes or 0.5 ml of every water sample in a 14 ml clean glass test tube for the MMS serie, 1 st line control and samples. Preparing of the MMS serie, 1st line control and samples.

Table 1: Preparing MMS Level MSS III (4.3.6) MSS II (4.3.4) ISS II (4.3.5) (µg/kg) 10 µg/l (µl) 100 µg/l (µl) 100 µg/l (µl)

MMS A 0 0 - 20 MMS B 0.2 10 - 20 MMS C 0.5 25 - 20 MMS D 1.0 50 - 20 MMS E 2.0 - 10 20 MMS F 5.0 - 25 20 1st Line control 2.0 - 10 20 Samples - - - 20

- For water samples proceed sample preparation after transferring supernatant of other matrices in a clean glass test tube. - Add 1 ml extraction solution acetonitrile:water (60:40 v/v%) (4.4.10). - The (water) samples are being processed with a bead ruptor to mechanically digest the meat and free the analytes from the cells. Place the tubes in the carriage kit and lock it. - Start program 3: speed 5.65 m/s, cycle time: 0.75 min, number of cycles: 2, pause dwell: 0.50 min. - Place the tubes in a centrifuge for 10 minutes at 3452 g. - Transfer the supernatant in a clean glass test tube (60% acetonitrile). Try to avoid transferring the fat layer as much as possible. Add 2 ml of water to dilute the supernatant. Add 0.75 ml of acetonitrile and 2.5 ml of water to dilute the water samples to a final content of 20% acetonitrile. Homogenize carefully. - Add 2 ml of water to dilute the supernatant to a final content of 20% acetonitrile. Add 0.75 ml of acetonitrile and 2.5 ml of water to dilute he water samples to a final content of 20% acetonitrile. Otherwise, the presence of a high concentration of acetonitrile will cause loss of analytes during application onto the Oasis HLB sorbent. - Continue 96-wells purification (6.5.2).

6.5.2 Sample clean-up by 96-wells SPE The 96-wells Oasis HLB plate is preconditioned with 1 ml of MeOH followed by 1 ml of water. Transfer all of the diluted supernatant of the sample obtained in 6.5.1 to the well in two portions (as 3 ml will not fit into a 2 ml well. E.g. 2 times 1.5 ml). Wash with 1 ml water and dry. Wash successively with wash solvent 1 to 6 (4.4.2 to 4.4.7). Dry after each wash step. Elute with 1 ml ACN. Collect the eluate in a 96-well collection plate in which already is 20 µl of DMSO pipetted. The eluate is evaporated till dryness under a gentle stream of nitrogen until only the DMSO is left. Add 50 µl of 10% MeOH and vortex carefully. The samples are now suitable for LC- MS/MS analysis. 10 µl of the mixture is injected directly into the UPLC-MS/MS system.

6.6 LC-MSMS analysis 6.6.1 LC-conditions

Inlet Method File: C:\masslynx\default.pro\acqudb\SOPA1242.acm

Analytical column: Acquity UPLC BEH C 18 100 mm x 1.0 mm, id 1.7 µm (5.16.2) Mobile phase A : Water/ACN/FA/ammonium formate 1M 900/100/0.02/2 (v/v/v/v) Mobile phase B: Water/ACN/FA/ammonium formate 1M 100/900/0.02/2 (v/v/v/v) Sample manager wash: ACN/water/FA 900/100/0.1 (v/v/v)

Sample manager purge: H2O/ACN 700/300 (v/v)

Seal wash: H2O/MeOH 900/100 (v/v) Column temperature: 60ºC Vial tray temperature: 20°C Injection volume: 10 µl (partial loop with needle overfill and load ahead) Flow: 0.15 ml/min

Table 2: Gradient profile for LC-MS/MS

Time Mobile phase A Mobile phase B Flow (min) (%) (%) (ml/min) 0.0 80 20 0.15 0.2 80 20 0.15 3.2 70 30 0.15 7.5 50 50 0.15 7.6 0 100 0.15 8.6 0 100 0.15 8.7 80 20 0.15 10.0 80 20 0.15

6.6.2 MSMS conditions (tune-file) Mass-spectrometric (MS) analysis is carried out on a Waters-XEVO-TQS UPLC-MSMS system (5.16) Parameter file: C:\MassLynx\Default.pro\Acqudb\SOPA1242.IPR

Table 3: Parameters for MSMS analysis

Parameter ESI ESI Positive Negative Capillair voltage 3.0 2.5 LM 1 resolution 3.0 2.8 HM 1 resolution 15.0 15.0 Ion energy 1 1.0 2.0 LM 2 resolution 3.0 3.0 HM 2 resolution 15.0 15.0 Ion energy 1 1.0 2.0 Source temperature 150°C Desolvation temperature 400°C Desolvation gas flow 800 l/hour Cone gas flow 150 l/hour Nebuliser gas flow 7 bar CID gas Argon, p=2,2*10-3 mbar, purity > 99.998% CID gas flow 0.18 ml/min

6.6.3 Regular MS-conditions The compounds fragment to structure related product ions. In table 4 the theoretical mono-isotopic masses of the precursor ions and corresponding product ions are displayed. For each of the mono-isotopic masses the permitted deviation is ± 0.5 Da. Parameter file: C:\MassLynx\Default.pro\Acqudb\SOP A1242.EXP

Table 4: Guidelines MS/MS fragmentation (mono-isotopic masses) Component I.S. Pre- Product Cone Collision ESI I.S. (component ID) Rt (min) ID cursor Ion Voltage energy + / - Ion (m/z) (m/z) (V) (eV) 17a/b-Trenbolone 271.20 199.10 30 25 + 1 3.56 / 3.10 253.20 30 20 + 17a/b-Trenbolone-d3 1 274.20 256.20 30 20 + 3.51 / 3.07 17a/b-Nortestosterone 275.20 109.10 30 25 + 2 4.71 / 3.64 145.10 30 20 + 17b-Nortestosterone-d3 2 278.20 109.10 30 25 + 3.67 17a/b-Boldenone 287.20 121.10 25 25 + 3 4.67 / 3.56 135.10 30 15 + 17a/b-Boldenone-d3 3 290.20 121.10 25 25 + 4.63 / 3.52 Methylboldenone 301.20 121.10 20 25 + 21 4.17 149.10 20 15 + Methylboldenone-d3 21 304.20 121.10 20 25 + 4.12 17a/b-Testosterone 289.20 97.10 30 25 + 4 5.50 / 4.45 109.10 30 25 +

Component I.S. Pre- Product Cone Collision ESI I.S. (component ID) Rt (min) ID cursor Ion Voltage energy + / - Ion (m/z) (m/z) (V) (eV) 17b-Testosterone-d3 4 292.20 97.10 30 25 + 4.41 17a-Methyltestosterone 303.20 97.10 30 25 + 5 5.12 109.10 30 30 + 17a-Methyltestosterone-d3 5 306.10 97.10 35 25 + 5.10 16b-OH-Stanozolol 345.20 81.10 30 45 + 6 3.04 95.10 30 40 + 16b-OH-Stanozolol-d3 6 348.25 81.10 50 45 + 3.02 Androstadienedione (ADD) 285.20 121.10 25 25 + 2 4.00 147.10 20 15 + Clobetasol 411.20 391.20 20 5 + 10 5.12 355.10 20 13 + Megestrolacetate (MGA) 385.24 325.20 35 13 + 7 7.54 267.20 35 18 + 7.52 Megestrolacetate-d3 (MGA-d3) 7 388.25 328.20 35 5 + Melengestrolacetate 397.24 337.20 35 13 + 8 7.88 279.10 35 20 + Melengestrolacetate-d3 8 400.20 337.20 35 5 + 7.86 Medroxyprogesterone-acetate 387.25 327.20 35 15 + 9 7.91 123.10 35 25 + Medroxyprogesterone-acetate-d3 (MPA- 7.89 d3) 9 390.25 330.20 35 5 + Chlormadinone-acetate (CMA) 405.18 345.20 35 13 + 7 7.84 309.10 35 15 + Triamcinolone-acetonide 435.30 415.30 40 8 + 10 3.28 339.20 40 13 + 3.25 Triamcinolone-acetonide-d6 10 441.30 421.30 40 8 + Zearalenone 317.20 175.08 44 24 - 11 5.47 317.20 131.15 44 30 - Zearalenone-d6 11 323.20 175.10 50 23 - 5.43 a/b-Zearalenol 319.20 275.20 30 21 - 12/13 4.33 / 3.34 160.15 30 30 - a/b-Zearalenol-d4 12/13 323.20 160.10 45 32 - 4.32 / 3.29 a/b-Zearalanol 321.20 277.20 56 20 - 14/15 4.09 / 3.18 259.20 56 26 - a/b-Zearalanol-d4 14/15 325.20 281.10 50 20 - 4.06 / 3.16 Zearalanone 319.20 275.20 30 21 - 16 5.39 205.20 54 22 - Zearalanone-d6 16 325.20 281.10 50 20 - 5.34 Dexamethasone*) / Betamethasone 437.20 307.00 20 30 - 17 2.80 / 2.75 361.10 20 15 - Dexamethasone-d4 *) 17 441.20 363.10 20 15 - 2.78 Prednisolone *) 405.20 329.10 20 15 - 18 1.87 280.10 20 35 - Prednisolone-d7 *) 18 412.20 336.13 20 15 - 1.80 Methylprednisolone *) 419.20 343.10 20 18 - 18 2.70

Component I.S. Pre- Product Cone Collision ESI I.S. (component ID) Rt (min) ID cursor Ion Voltage energy + / - Ion (m/z) (m/z) (V) (eV)

Flumethasone *) 455.20 379.10 20 20 - 17 2.78 305.00 20 30 - Isoflupredone *) 423.20 347.10 20 15 - 19 1.81 293.00 20 30 - Isoflupredone-d3 *) 19 426.20 350.10 20 17 - 1.78 Stanozolol 329.20 81.10 20 40 + 20 5.55 121.10 20 40 + Stanozolol-d3 20 332.20 81.10 20 40 + 5.53 *) Formic acid adduct (FA adduct)

6.6.4 Initial test LC system By means of MSS II (4.3.4) the retention time, sensitivity and stability of the system are tested. Calculate the signal/noise ratio of the lowest ion of each compound. The signal/noise ratio should be at least 6 for each compound.

6.6.5 Sequence analysis sample series After the initial test the sequence the recommended order of analysis is: - MMS series - 1st line control - Blanc solvent - Samples - Blanc solvent - MMS series

7 RESULTS 7.1 Calculations Table 4 is for each compound defined which internal standard is used for calculations.

Calculations are performed by Quanlynx v4.1 (Waters). With QuanLynx software, you quantitate compounds for many different types of application. Calibration curves are generated using standard samples containing compounds of known concentrations. The calibration curves are then used to calculate the concentrations of compounds in analyte samples. You specify a list of samples and a quantify method that describes how to process each of the compounds in these samples. See appendix 4. The following equations are automatically applied within Quanlynx:

Equation I: Calculation response factor (RF)

 Area   compound  RF =    Area IS  With: RF = response factor

Area compound = sum area product ions

Area IS = area product ion internal standard

Equation II: Calculation of the concentration in the sample (X)

 RF − b  X =    a 

With: X = concentration of a compound in the sample (µg/kg) RF = response factor (equation I (7.1)) . b = intersection point of the calibration curve with the y-axis (follows from linear regression*) a = slope of the calibration line (follows from linear regression*) *Plot the area of the MMS measured before and after the sample extracts as function of the added levels. The linear regression is calculated using the 1/X weighting function.

Equation III: Calculation relative retention time (RRT)

 RT  = compound RRT    RT IS  With: RRT = relative retention time

RT compound = retention time of compound

RT IS = retention time of internal standard 7.2 Criteria 7.2.1 General For acceptance of the analysis results, the performance criteria (stated below) have to be met. The concentrations are automatically calculated by Quanlynx software.

7.2.2 Linearity The MMS are used to determine the linearity of the LC-MS/MS system and to determine if the sample pre- treatment is done correctly. The correlation of the line should be ≥ 0.990.

7.2.3 Sensitivity The S/N of the most intense product ion of MMS 0.25*RW should be ≥ 6.

7.2.4 Accuracy Calculate the concentration in the 1 st line control using equation II. Calculate the accuracy for the selected compounds by dividing the concentration by the actual spiked level (%). Fill in the Shewart charts. SOP F0057 discusses Shewhart charts. Use a sample that already proved to be blanc. Be aware for natural occurring compounds like testosterone, nortestosterone, boldenone and RALs. Shewhart charts are available for the next components in muscle, poultry liver, water for poultry and fish samples: Muscle: nortestosterone-17 β, stanozolol, chlormadinone-acetate, zearalanol-α, beta-&dexamethasone Poultry liver: methylboldenone-17 α, stanozolol, melengestrol-acetate, zearalenone, clobetasol Water for poultry: methyltestosterone-17 α, stanozolol, megestrol-acetate, zearalanone, flumethasone Fish: testosteron-17 β, boldenone-17 α, medroxyprogesterone-acetate, zearalanol-β, triamcinolone-acetate

7.2.5 Maximum deviation retention time The expected retention time f each component at any sample is based on the retention time of the related internal standard (see table 4). At this way any shift in retention time will be adjusted and shown as a predicted retention time for each component at any time.

7.2.6 Quantification The concentration of the compounds in the sample is expressed as µg/kg and is calculated using the linear regression curves calculated from the MMS series. Calculate the concentrations using equations I and II (7.1). The concentration is automatically calculated using Quanlynx (appendix 4).

7.2.7 Criteria for individual samples Calculate for each sample the relative deviation of the retention time. When the relative deviation of the retention time does not exceed 2.5% and the concentration exceeds CC α, the sample is suspect. The second and/or third validation level decides whether the sample is reanalysed for confirmatory analysis according to CD 2002/657.

7.3 Final results When a compound is identified in the sample with a concentration exceeding CC α and is confirmed according to CD 2002/657, the concentration is reported in LIMS. When no compound is identified in the sample the CC α is reported in LIMS.

Table 5: Registration in LIMS when “not confirmed”

Component Bovine Other Poultry Water for Fish meat meat Liver Poultry (µg/kg) (µg/kg) (µg/kg) (µg/kg) (µg/kg) 17 α-boldenone <0.41 <2 <2 <2 <2 17 β-boldenone <0.61 <2 <2 <2 <2 17 α-methyltestosterone <0.69 <2 <2 <2 <2 17 α-nortestosterone <0.73 <2 <2 <2 <2 17 β-nortestosterone <0.54 <2 <2 <2 <2 17 α-testosterone <1.42 <2 <2 <2 <2 17 β-testosterone <0.85 <2 <2 <2 <2 17 α-trenbolone <0.42 <2 <2 <2 <2 17 β-trenbolone <0.32 <2 <2 <2 <2 androsta-1,4-diene-3,17-dione (ADD) <0.60 <2 <2 <2 <2 methylboldenone <0.80 <2 <2 <2 <2 stanozolol <2.86 <2 <2 <2 <2 16 β-OH-stanozolol <0.31 <2 <2 <2 <2 prednisolone <1 <2 <2 <2 <2 methylprednisolone <1 <2 <2 <2 <2 isoflupredon <1 <2 <2 <2 <2 flumethason <1.03 <2 <2 <2 <2 dexamethasone *) <1.89 <4 <4 <4 <4 betamethasone *) <1.89 <4 <4 <4 <4 triamcinolon-acetonide <0.29 <2 <2 <2 <2 clobetasol <1.94 <2 <2 <2 <2 megestrol-acetate <1.26 <2 <2 <2 <2 melengestrol-acetate <0.26 <2 <2 <2 <2 medroxyprogesterone-acetate <0.80 <2 <2 <2 <2 chlormadinone-acetate <1.43 <2 <2 <2 <2 α-zeranol ( α-zearalanol) <2.42 <2 <2 <2 <2 β-zeranol ( β-zearalanol) <2.80 <2 <2 <2 <2 zearalanon <1 <2 <2 <2 <2 zearalenon <1.98 <2 <2 <2 <2 α-zearalenol <1 <2 <2 <2 <2 β-zearalenol <1 <2 <2 <2 <2 Bold : cc β (no cc α available) *) Sum of beta- and dexamethason

8 REGISTRATION

The MS/MS data are saved in a directory on the computer. All information concerning the analysis is described in the labjournal.

LITERATURE

[1] EU Beslissing 2002/657/EC, J.EUR. Communities, L221 (2002) 8 [2] SOP F0057 – Controle kaarten – Gebruik en beheer [3] SOP F0071 – Persoonlijke beschermingsmiddelen binnen het RIKILT

Appendix 1a belonging to SOP A1242 Performance sheet Bovine muscle, 3-day validation

Analyt

* (%) * / / ) r -%) -%) -% RL

α β Rel st. dev. st.dev. Rel Repeatability Concentration Concentration range / interval Unit Accuracy Trueness accuracy Rel.st.dev. (RSD (RSDr CC CC 17b-Testosterone 1.0 µg/kg 101 13.0 13.0 0.85 1.70 2.0 µg/kg 99 14.5 12.8 3.0 µg/kg 96 17.3 6.9

17b -Nortestosterone 1.0 µg/kg 101 5.0 4.9 0.54 1.08 2.0 µg/kg 98 6.6 4.7 3.0 µg/kg 98 9.2 5.7

17b-Boldenone 1.0 µg/kg 100 9.2 8.0 0.61 1.23 2.0 µg/kg 99 9.0 4.9 3.0 µg/kg 98 9.6 5.5

17a -Trenbolone 1.0 µg/kg 98 10.1 9.4 0.42 0.83 2.0 µg/kg 97 5.6 5.0 3.0 µg/kg 99 3.9 3.7

17a-Testosterone 1.0 µg/kg 96 9.4 9.0 1.42 2.84 2.0 µg/kg 91 11.4 7.3 3.0 µg/kg 87 19.6 10.7

17a-Nortestosterone 1.0 µg/kg 102 5.2 5.1 0.73 1.46 2.0 µg/kg 102 9.4 9.2 3.0 µg/kg 99 8.5 8.2

17a -Boldenone 1.0 µg/kg 102 9.0 5.7 0.41 0.82 2.0 µg/kg 102 10.4 5.3 3.0 µg/kg 105 4.6 4.5

17b-Trenbolone 1.0 µg/kg 100 5.4 5.3 0.32 0.64 2.0 µg/kg 97 5.2 4.8 3.0 µg/kg 98 4.6 3.9

16b-OH-Stanozolol 1.0 µg/kg 100 9.0 7.7 0.31 0.63 2.0 µg/kg 100 4.8 4.4 3.0 µg/kg 102 4.1 2.9

a-Zearalanol 1.0 µg/kg 103 14.6 14.5 1.21 2.42 2.0 µg/kg 99 14.6 14.0 3.0 µg/kg 102 12.9 12.4

Androstadiendione 1.0 µg/kg 98 8.3 7.8 0.60 1.21 2.0 µg/kg 99 6.4 5.8 3.0 µg/kg 100 7.2 6.9

b-Zearalenol 1.0 µg/kg 99 * * <1 1 2.0 µg/kg 105 * * 3.0 µg/kg 104 * *

b-Zearalanol 1.0 µg/kg 98 * * <1 1 2.0 µg/kg 92 * * 3.0 µg/kg 96 13.6 13.0

a-Zearalenol 1.0 µg/kg 77 * * <1 1 2.0 µg/kg 73 * * 3.0 µg/kg 83 * *

Clobetasol 1.0 µg/kg 96 * * 0.97 1.94 2.0 µg/kg 97 10.4 10.0

3.0 µg/kg 104 17.3 14.8

Chlormadinone-acetate 1.0 µg/kg 103 19.1 1.43 2.86 2.0 µg/kg 99 13.3 12.1 3.0 µg/kg 99 16.6 9.2

Beta - + Dexamethason (ESI+) 2.0 µg/kg 104 9.5 9.1 1.89 3.78 4.0 µg/kg 100 11.1 10.5 6.0 µg/kg 102 11.0 10.3

Flumethason-FA adduct 1.0 µg/kg 99 10.7 9.6 1.03 2.05 2.0 µg/kg 100 15.2 13.4 3.0 µg/kg 92 13.2 12.7

Isoflupredon-FA adduct 1.0 µg/kg 125 * * <1 1 2.0 µg/kg 114 * * 3.0 µg/kg 134 * *

Megestrolacetate 1.0 µg/kg 104 18.7 1.26 2.51 2.0 µg/kg 101 15.3 10.3 3.0 µg/kg 104 12.8 9.6

Medroxyprogesterone-acetate 1.0 µg/kg 107 10.7 6.3 0.80 1.59 2.0 µg/kg 102 14.8 8.2 3.0 µg/kg 103 10.5 6.7

Triamcinolonacetonide 1.0 µg/kg 102 8.3 7.1 0.29 0.58 2.0 µg/kg 101 6.1 4.1 3.0 µg/kg 99 4.5 3.5

Prednisolone-FA adduct 1.0 µg/kg 115 * * <1 1 2.0 µg/kg 109 * * 3.0 µg/kg 82 * *

* * Stanozolol (incl. interferentie op 1.0 µg/kg 134 <1 1 meest intense overgang) 2.0 µg/kg 102 16.6 3.0 µg/kg 97 11.4 10.9

Melengestrolacetate 1.0 µg/kg 102 4.5 4.2 0.26 0.51 2.0 µg/kg 99 7.3 3.9 3.0 µg/kg 100 4.6 2.7

Methylprednisolone-FA adduct 1.0 µg/kg 89 * * <1 1 2.0 µg/kg 84 * * 3.0 µg/kg 81 * *

Methylboldenone 1.0 µg/kg 101 20.3 13.7 0.80 1.60 2.0 µg/kg 102 15.1 8.3 3.0 µg/kg 101 18.8 11.4

Methyltestosterone 1.0 µg/kg 100 8.7 8.2 0.69 1.38 2.0 µg/kg 99 5.6 5.0 3.0 µg/kg 100 8.2 7.7

Zearalanon 1.0 µg/kg 99 * * <1 <1 2.0 µg/kg 94 19.7 * 3.0 µg/kg 96 * *

Zearalenon 1.0 µg/kg 102 11.1 9.9 0.99 1.98 2.0 µg/kg 101 12.0 11.9 3.0 µg/kg 98 11.6 9.5

* Can not be confirmed and determined at specified concentration. Can only either be identified, determined or confirmed.

Appendix 1b belonging to SOP A1242 Performance sheet Muscle (other than bovine), 1-day validation

Analyt

* (%) * ) r -%) -%) -% RL

α β Rel st. dev. st.dev. Rel Repeatability Concentration Concentration range / interval Unit / Accuracy Trueness accuracy Rel.st.dev. (RSD (RSDr CC CC 17b-Testosterone 2.0 µg/kg 100 9.6 6.0 <2 2

17b-Nortestosterone 2.0 µg/kg 84 13.4 8.4 <2 2

17b -Boldenone 2.0 µg/kg 90 15.4 9.7 <2 2

17a -Trenbolone 2.0 µg/kg 94 5.3 3.3 <2 2

17a-Testosterone 2.0 µg/kg 89 11.7 7.3 <2 2

17a-Nortestosterone 2.0 µg/kg 106 9.2 5.7 <2 2

17a -Boldenone 2.0 µg/kg 102 20.5 12.8 <2 2

17b-Trenbolone 2.0 µg/kg 92 8.3 5.2 <2 2

16b-OH-Stanozolol 2.0 µg/kg 98 4.9 3.0 <2 2

a-Zearalanol 2.0 µg/k g 99 7.3 4.6 <2 2

Androstadiendione 2.0 µg/kg 83 10.9 6.8 <2 2

b-Zearalenol 2.0 µg/kg * * * - 2

b-Zearalanol 2.0 µg/kg * * * - 2

a-Zearalenol 2.0 µg/kg * * * - 2

Clobetasol 2.0 µg/kg * * * - 2

Ch lormadinone -acetate 2.0 µg/kg 92 9.2 5.7 <2 2

Beta- + Dexamethason (ESI+) 2.0 µg/kg 97 3.1 1.9 <2 2

Flumethason-FA adduct 2.0 µg/kg * * * - 2

Isoflupredon -FA adduct 2.0 µg/kg * * * - 2

Megestrolacetate 2.0 µg/kg 86 8.7 5.4 <2 2

Medroxyprogesterone-acetate 2.0 µg/kg 87 5.9 3.7 <2 2

Triamcinolonacetonide 2.0 µg/kg 98 3.1 1.9 <2 2

Prednisolone -FA adduct 2.0 µg/kg * * * - 2

Stanozolol (incl. interferentie op 2.0 µg/kg 86 12.7 8.0 <2 2 meest intense overgang)

Melengestrolacetate 2.0 µg/kg 93 3.4 2.1 <2 2

Methylprednisolone-FA adduct 2.0 µg/kg * * * - 2

Methylboldenone 2.0 µg/kg 91 6.0 3.8 <2 2

Methyltestosterone 2.0 µg/kg 95 13.4 8.4 <2 2

Zearalanon 2.0 µg/kg 99 21.6 13.5 <2 2

Zearalenon 2.0 µg/kg 97 9.6 6.0 <2 2

* Can not be confirmed and determined at specified concentration. Can only either be identified, determined or confirmed.

Appendix 1c belonging to SOP A1242 Performance sheet Poultry liver, 1-day validation

Analyt

* (%) * ) ) r -%) -%) -% RL

α β Rel st. dev. st. dev. Rel Repeatability Concentration Concentration range / interval Unit / Accuracy Trueness accuracy Rel.st.dev. (RSD (RSDr CC CC 17b-Testosterone 2.0 µg/kg 103 * * - 2

17b -Nortestosterone 2.0 µg/kg * * * - 2

17b-Boldenone 2.0 µg/kg * * * - 2

17a-Trenbolone 2.0 µg/kg 104 14.1 8.8 <2 2

17a -Testosterone 2.0 µg/kg * * * - 2

17a-Nortestosterone 2.0 µg/kg 108 * 14.5 - 2

17a-Boldenone 2.0 µg/kg 109 21.8 13.6 <2 2

17b -Trenbolone 2.0 µg/kg 95 10.2 6.4 <2 2

16b-OH-Stanozolol 2.0 µg/kg 106 6.9 4.3 <2 2

a-Zearalanol 2.0 µg/kg * * * - 2

Androstadiendione 2.0 µg/kg * * * - 2

b-Zearalenol 2.0 µg/kg * * * - 2

b-Zearalanol 2.0 µg/kg * * * - 2

a-Zearalenol 2.0 µg/kg * * * - 2

Clobetasol 2.0 µg/kg * 21.8 13.6 <2 2

Chlormadinone-acetate 2.0 µg/kg * * * - 2

Beta- + Dexamethason (ESI+) 2.0 µg/kg 99 14.6 9.1 <2 2

Flumethason -FA adduct 2.0 µg/kg * * * <2 2

Isoflupredon-FA adduct 2.0 µg/kg * * * <2 2

Megestrolacetate 2.0 µg/kg * * * <2 2

Medroxyprogesterone -acetate 2.0 µg/kg 85 17.4 10.9 2.49 2.97

Triamcinolonacetonide 2.0 µg/kg 96 10.4 6.5 2.33 2.65

Prednisolone-FA adduct 2.0 µg/kg * * * <2 2

Stanozolol (incl. interferentie op 2.0 µg/kg 85 10.4 6.5 <2 2 meest intense overgang)

Melengestrolacetate 2.0 µg/kg 98 5.6 3.5 <2 2

Methylprednisolone-FA adduct 2.0 µg/kg * * * - 2

Methylboldenone 2.0 µg/kg 105 19.7 12.3 <2 2

Methyltestosterone 2.0 µg/kg * * * - 2

Zearalanon 2.0 µg/kg * * * - 2

Zearalenon 2.0 µg/kg 91 11.9 7.5 <2 2

* Can not be confirmed and determined at specified concentration. Can only either be identified, determined or confirmed.

Appendix 1d belonging to SOP A1242 Performance sheet Animal tap water, 1-day validation

Analyt

* (%) * ) r -%) -%) -% al / range range al/ RL

α β Rel st. dev. st.dev. Rel Repeatability Concentration Concentration interv Unit / Accuracy Trueness accuracy Rel.st.dev. (RSD (RSDr CC CC 17b-Testosterone 2.0 µg/kg 101 2.8 1.8 <2 2

17b-Nortestosterone 2.0 µg/kg 101 3.4 2.1 <2 2

17b -Boldenone 2.0 µg/kg 101 3.4 2.1 <2 2

17a -Trenbolone 2.0 µg/kg 104 2.6 1.7 <2 2

17a-Testosterone 2.0 µg/kg 102 2.8 1.8 <2 2

17a-Nortestosterone 2.0 µg/kg 103 4.1 2.5 <2 2

17a -Boldenone 2.0 µg/kg 102 4.4 2.7 <2 2

17b-Trenbolone 2.0 µg/kg 102 3.6 2.2 <2 2

16b-OH-Stanozolol 2.0 µg/kg 104 3.8 2.4 <2 2

a-Zearalanol 2.0 µg/ kg * 20.7 13 - 2

Androstadiendione 2.0 µg/kg 105 3.4 2.1 <2 2

b-Zearalenol 2.0 µg/kg * 19.1 11.9 - 2

b-Zearalanol 2.0 µg/kg * 14.1 8.8 - 2

a-Zearalenol 2.0 µg/kg * * * - 2

Clobetasol 2.0 µg/kg 95 * * - 2

Chlormadinone -acetate 2.0 µg/kg 106 9.9 6.2 <2 2

Beta- + Dexamethason (ESI+) 2.0 µg/kg 100 7.7 4.8 <2 2

Flumethason-FA adduct 2.0 µg/kg 99 9.8 6.1 <2 2

Isoflupredon -FA adduct 2.0 µg/kg * * * - 2

Megestrolace tate 2.0 µg/kg 104 13.1 8.2 <2 2

Medroxyprogesterone-acetate 2.0 µg/kg 103 2.5 1.6 <2 2 (MPA)

Triamcinolonacetonide 2.0 µg/kg 103 3.7 2.3 <2 2

Prednisolone -FA adduct 2.0 µg/kg * * * - 2

Stanozolol (incl. interferentie op 2.0 µg/kg 97 3.5 2.2 <2 2 meest intense overgang)

Melengestrolacetate 2.0 µg/kg 102 5.6 3.5 <2 2

Methylprednisolone -FA adduct 2.0 µg/kg * * * - 2

Methylboldenone 2.0 µg/kg 103 7.9 5.0 <2 2

Methyltestosterone 2.0 µg/kg 103 4.0 2.5 <2 2

Zearalanon 2.0 µg/kg * 17.3 10.8 - 2

Zearalenon 2.0 µg/kg * 16.3 10.2 - 2

* Can not be confirmed and determined at specified concentration. Can only either be identified, determined or confirmed.

Appendix 1e belonging to SOP A1242 Performance sheet Fish (unspecified, different species) , 1-day validation

Analyt

* (%) * ) r -%) -%) -% RL

α β Rel st. dev. st.dev. Rel Repeatability Concentration Concentration range / interval Unit / Accuracy Trueness accuracy Rel.st.dev. (RSD (RSDr CC CC 17b-Testosterone 2.0 µg/kg 98 10.1 6.3 <2 2

17b-Nortestosterone 2.0 µg/kg 94 21.9 13.7 <2 2

17b -Boldenone 2.0 µg/kg 103 15.1 9.5 <2 2

17a -Trenbolone 2.0 µg/kg 91 11.1 6.9 <2 2

17a-Testosterone 2.0 µg/kg 106 21.3 13.3 <2 2

17a-Nortestosterone 2.0 µg/kg 99 4.4 2.7 <2 2

17a -Boldenone 2.0 µg/kg 98 7.0 4.4 <2 2

17b-Trenbolone 2.0 µg/kg 90 13.4 8.4 <2 2

16b-OH-Stanozolol 2.0 µg/kg 105 5.5 3.4 <2 2

a-Zearalanol 2.0 µg/kg 100 15.2 9.5 <2 2

Androstadiendione 2.0 µg/kg 96 23.1 14.5 <2 2

b-Zearalenol 2.0 µg/kg * * * - 2

b-Zearalanol 2.0 µg/kg * * * - 2

a-Zearalenol 2.0 µg/kg 104 * * - 2

Clobetasol 2.0 µg/kg * * * - 2

Chl ormadinone -acetate 2.0 µg/kg 96 17.5 10.9 <2 2

Beta- + Dexamethason (ESI+) 2.0 µg/kg * 8.0 5.0 - 2

Flumethason-FA adduct 2.0 µg/kg * * 14.6 - 2

Isoflupredon -FA adduct 2.0 µg/kg 73 * * - 2

Megestrolacetate 2.0 µg/kg 90 22.0 13.7 <2 2

Medroxyprogesterone-acetate 2.0 µg/kg 87 17.5 10.9 <2 2 (MPA)

Triamcinolonacetonide 2.0 µg/kg 91 11.3 7.1 <2 2

Prednisolone -FA adduct 2.0 µg/kg * * * - 2

Stanozolol (incl. interferentie op 2.0 µg/kg 83 * * - 2 meest intense overgang)

Melengestrolacetate 2.0 µg/kg 97 5.2 3.2 <2 2

Methylprednisolone -FA adduct 2.0 µg/kg * * * - 2

Methylboldenone 2.0 µg/kg 106 10.7 6.7 <2 2

Methyltestosterone 2.0 µg/kg 105 21.8 13.6 <2 2

Zearalanon 2.0 µg/kg * * - 2

Zearalenon 2.0 µg/kg 100 * * - 2

* Can not be confirmed and determined at specified concentration. Can only either be identified, determined or confirmed.

Appendix 2 belonging to SOP A1242 Checklist

Date: ______Name analist: ______Labjournal/page: ______Working list number(s) ______

□ Add a single layer of beads (5.5) 7 ml test tubes) (5.4). □ Weigh 1 test unit (0.5 g) of every homogenized sample in 7 ml polypropylene tubes or 0.5 ml of every water sample in a 14 ml clean glass test tube (MMS, 1 st line control and test samples). □ Preparing of the MMS serie, 1st line control and samples. Level MSS III (4.3.6) MSS II (4.3.4) ISS II (4.3.5) (µg/kg) 10 µg/l (µl) 100 µg/l (µl) 100 µg/l (µl) MMS A 0 0 - 20 MMS B 0.2 10 - 20 MMS C 0.5 25 - 20 MMS D 1.0 50 - 20 MMS E 2.0 - 10 20 MMS F 5.0 - 25 20 1st Line control 2.0 - 10 20 Samples - - - 20

□ For water samples proceed sample preparation after transfering supernatant of other matrices in a clean glass test tube. □ Add 1 ml extraction solution acetonitrile:water (60:40 v/v%) (4.4.10) to the samples in the 7 ml polypropylene tubes. □ Process MMS, 1 st line control and samples with the bead ruptor (5.3). Program 3: speed 5.65 m/s, cycle time: 0.75 min, number of cycles: 2, pause dwell: 0.50 min. □ Place the tubes in a centrifuge for 10 minutes at 3452 g. □ Transfer the supernatant in a clean glass test tube. Try to avoid transferring the fat layer as much as possible. □ Add 2 ml of water to dilute the supernatant. Add 0.75 ml of acetonitrile and 2.5 ml of water to dilute the water samples to a final content of 20% acetonitrile. Homogenize carefully. □ Pre-condition the 96 wells SPE plate with 1 ml of MeOH followed by 1 ml of water. □ Bring the whole sample into the SPE well in portions (e.g. 2 times 1,5 ml). □ Wash with 1 ml of water and dry. □ Wash with 1 ml wash solvent 1 (4.4.2) and dry. □ Wash with 1 ml wash solvent 2 (4.4.3) and dry. □ Wash with 1 ml wash solvent 3 (4.4.4) and dry.

□ Wash with 1 ml wash solvent 4 (4.4.5) and dry. □ Wash with 1 ml wash solvent 5 (4.4.6) and dry. □ Wash with 1 ml wash solvent 6 (4.4.7) and dry. □ Pipet 20 µl DMSO in each well of the collection plate. □ Elute with 1 ml ACN. □ Place plate in a 96-wells evaporator and dry till only DMSO is left. □ Add 50 µl of 10% MeOH and vortex carefully. □ The samples are now suitable for LC-MS/MS analysis.

Appendix 3 belonging to SOP A1242 EXACT MASS OF ALL COMPOUNDS FOR HIGH RESOLUTION MS

The detection of the extracts obtained with this method can also be analysed by use of high resolution mass spectrometry. The following settings have to be used. The LC-HRMS system consisted of an Ultimate 3000 LC system coupled through a HESI II electrospray source to an Q-Exactive Orbitrap MS (Thermo Fisher Scientific, San Jose, CA, USA). The same column and gradient was used as on the triple quad system. The electrospray source was operated in alternating positive and negative ionization mode and the electrospray settings were as follows: electrospray voltage, 3.5 kV; sheath gas, 47.5 AU; auxiliary gas, 11.5 AU. The probe-heater was set to 410°C, and the heated capillary was set at 320°C. Data were acquired by full-scan (135-1000 amu) measurement. The resolving power for the full-scan event was 70,000, defined at full width half maximum at m/z 200. The other parameters for the mass spectrometer were automatically tuned with the tuning and calibration procedure. Before analysis, the mass calibration of the mass spectrometer was checked and re-calibrated if needed.

Exact Mass Exact Mass Exact Mass Component Formula M+H M-H M+FA-H 16b-OH Stanozolol C21H32N2O2 345.25365 16b -OH Stanozolol -d3 C21H29N2O2D3 348.27249 Stanozolol C21H32N2O 329.25874 Stanozolol-d3 C21H29N2OD3 332.27757 17a -Boldenone C19H26O2 287.20056 17a-Boldenone-d3 C19H23O2D3 290.21939 17b -Boldenone C19H26O2 287.20056 17b -Boldenone -d3 C19H23O2D3 290.21939 Methylboldenone C20H28O2 301.21621 Methylboldenone -d3 C20H25O2D3 304.23504 17a -MTT C20H30O2 303.23186 17a-MTT-d3 C20H27O2D3 306.25069 17a -NT C18H26O2 275.20056 17b-NT C18H26O2 275.20056 17b-NT-d3 C18H23O2D3 278.21939 17a -Trenbolone C18H22O2 271.16926 17a-Trenbolone-d3 C18H19O2D3 274.18809 17b -Trenbolone C18H22O2 271.16926 17b -Trenbolone -d3 C18H19O2D3 274.18809 Androstadienedione C19H24O2 285.18491 17a -Testosterone C19H28O2 289.21621 17b -Testosterone C19H28O2 289.21621 17b-Testosterone-d3 C19H25O2D3 292.23504 a-Zearalanol C18H26O5 321.17075 a-Zearalanol-d4 C18H22O5D4 325.19585 b-Zearalanol C18H26O5 321.17075 b-Zearalanol -d4 C18H22O5D4 325.19585

a-Zearalenol C18H24O5 319.15510 a-Zearalenol-d4 C18H20O5D4 323.18020 b-Zearalenol C18H24O5 319.15510 b-Zearalenol-d4 C18H20O5D4 323.18020 Zearalanon C18H24O5 319.15510 Zearalanon -d6 C18H18O5 D6 325.19276 Zearalenon C18H22O5 317.13945 Zearalenon -d6 C18H16O5 D6 323.17711 Clobetasol C22H28ClFO4 411.17329 455.16422 Dexamethasone C22H29FO5 393.20718 437.19810 Dexamethasone -d4 C22H25FO5D4 397.23229 441.22321 Betamethasone C22H29FO5 393.20718 437.19810 Flumethasone C22H28F2O5 411.19776 455.18868 Isoflupredon C21H27FO5 379.19153 423.18245 Isoflupredon-d3 C21H24FO5D3 382.21036 426.20128 Methylprednisolone C22H30O5 375.21660 419.20753 Prednisolone C21H28O5 361.20095 405.19188 Prednisolone-d7 C21H21O5D7 368.24489 412.23581 Prednisolone-d8 C21H20O5D8 369.25116 413.24209 Triamcinolone -acetonide C24H31FO6 435.21774 479.20867 Triamcinolone-acetonide-d6 C24H25FO6D6 441.25540 485.24633 MGA C24H32O4 385.23734 MGA -d3 C24H29O4D3 388.25617 MLA C25H32O4 397.23734 MLA -d3 C25H29O4D3 400.25617 MPA C24H34O4 387.25299 MPA-d3 C24H31O4D3 390.27182 CMA C23H29ClO4 405.18271

Appendix 4 belonging to SOP A1242 Example of Quanlynx sequence Besides columns ‘MS Tune File’, ‘Inlet File’, ‘Bottle’ and ‘Inject Volume’, the columns ‘Sample Type’ and ‘Conc. A’ have to be filled in to use the quantification function in Quanlynx software.

File Name FileText Sample Conc. Type A

TQS1_170314_validatie_037 Blanco 20% dmso + 30% 10% MeOH Blank

TQS1_170314_validatie_048 MMS A 0 RW (0 ppb) vlees nr. 1 Standard 0.0

TQS1_170314_validatie_049 MMS B 0.2 RW (0.4 ppb) vlees nr. 1 Standard 0.4

TQS1_170314_validatie_050 MMS C 0.5 RW (1.0 ppb) vlees nr. 1 Standard 1.0

TQS1_170314_validatie_051 MMS D 1.0 RW (2.0 ppb) vlees nr. 1 Standard 2.0

TQS1_170314_validatie_052 MMS E 2.5 RW (5.0 ppb) vlees nr. 1 Standard 5.0

TQS1_170314_validatie_053 MMS F 5.0 RW (10.0 ppb) vlees nr. 1 Standard 10.0

TQS1_170314_validatie_054 Blanco 20% dmso + 30% 10% MeOH Blank

TQS1_170314_validatie_008 Recovery 1.0 RW (spike na opwerking) vlees nr. 1 Recovery 2.0

TQS1_170314_validatie_009 Blanco vlees nr. 2 Analyte

TQS1_170314_validatie_010 Blanco vlees nr. 3 Analyte

TQS1_170314_validatie_011 Blanco vlees nr. 4 Analyte

TQS1_170314_validatie_012 Blanco vlees nr. 5 Analyte

TQS1_170314_validatie_013 Blanco vlees nr. 6 Analyte

TQS1_170314_validatie_014 Blanco vlees nr. 7 Analyte

TQS1_170314_validatie_015 Blanco vlees nr. 8 Analyte

TQS1_170314_validatie_016 0.5 RW vlees nr. 2 Analyte

TQS1_170314_validatie_017 0.5 RW vlees nr. 3 Analyte

TQS1_170314_validatie_018 0.5 RW vlees nr. 4 Analyte

TQS1_170314_validatie_019 0.5 RW vlees nr. 5 Analyte

TQS1_170314_validatie_020 0.5 RW vlees nr. 6 Analyte

TQS1_170314_validatie_021 0.5 RW vlees nr. 7 Analyte

TQS1_170314_validatie_022 0.5 RW vlees nr. 8 Analyte

TQS1_170314_validatie_023 1.0 RW vlees nr. 2 Analyte

TQS1_170314_validatie_024 1.0 RW vlees nr. 3 Analyte

TQS1_170314_validatie_025 1.0 RW vlees nr. 4 Analyte

TQS1_170314_validatie_026 1.0 RW vlees nr. 5 Analyte

TQS1_170314_validatie_027 1.0 RW vlees nr. 6 Analyte

TQS1_170314_validatie_028 1.0 RW vlees nr. 7 Analyte

TQS1_170314_validatie_029 1.0 RW vlees nr. 8 Analyte

TQS1_170314_validatie_030 1.5 RW vlees nr. 2 Analyte

TQS1_170314_validatie_031 1.5 RW vlees nr. 3 Analyte

TQS1_170314_validatie_032 1.5 RW vlees nr. 4 Analyte

TQS1_170314_validatie_033 1.5 RW vlees nr. 5 Analyte

TQS1_170314_validatie_034 1.5 RW vlees nr. 6 Analyte

TQS1_170314_validatie_035 1.5 RW vlees nr. 7 Analyte

TQS1_170314_validatie_036 1.5 RW vlees nr. 8 Analyte

TQS1_170314_validatie_055 Recovery 1.0 RW (spike na opwerking) vlees nr. 1 Recovery 2.0

A Quanlynx method which can be used to process the obtained data see method:

SOP-A1242_TQS1_ RIK-LC-xxx.mdb, whereby xxx is the analytical column number according to RIKILT system.

The following Quanlynx settings are used:

- Conc. of Std: Levels Conc A and sample type, see above sequence table - Response uses: Area - Polynomial type: Linear - Calibration origin: Exclude - Weighting function: None - Concentration: ppb (ng/g) - Smooth enabled: YES - Smooth method: MEAN (Smooth iterations: 2 and Smooth width: 4)