Analytisch laboratorium voor voedings- en residue onderzoek SOP ARO/515 v 1

Printdatum: 1-Oct-08 r

Analysis of special in bovine urine by GC-MS/MS

Document: SOP ARO/515 versie 1 Auteur: Hennie van Rossum Documentgebied: ARO Methods Autorisator: Leen van Ginkel (labhoofd) Status: Definitief

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Printdatum: 1-Oct-08 r 1 Introduction This procedure describes a method for the screening of dromostanolone, methenolone, , , oxymesterone, chloromethylboldenone, and 16-hydroxyfurazabol in samples of urine of bovine origin. -d3 is used as internal standard. A combination of two solid phase extractions (SPE) and gas chromatography-mass spectrometry (GC-MS) is used. The method comprises the following steps: deconjugation of the steroids by an enzymatic hydrolysis. Purification is performed by SPE C18, LLE, and SPE OASIS. Detection as TMS derivative is performed with GC-MSMS, Electron Impact (EI).

2 Apparatus The GC-MS conditions in Table 1 are used. Table 1. Measurements parameters GC-MS. Carrier gas: 1.1 ml/min (constant flow) helium Column VF-17-MS 30 meter; 0.25 mm i.d.; 0.25 μm film. Injection 2 µl pulsed splitless (Injector temperature 250 volume °C) Detector 200 °C EI-source. temperature Interface 280 °C. temperature Oven 110 °C for 1 min, increased at 20°C/min. to temperature 280 °C and held at this temp. for 0,5 min. Then increased at 5°C/min. to 300 °C, after this a post-column-oven temperature at 340 °C for 4 minutes. Ionization Electron Impact Estradiol-d3 419>285 419>329 (-28Volt) (-15Volt) Dromostanolone 448>405 448>209 (-19Volt) (-30Volt) Methenolone 446>208 446>195 (-20Volt) (-30Volt) Mibolerone 446>431 446>341 (-15Volt) (- 30Volt). MRM’s Calusterone 460>445 460>315 measured (-20Volt) (-30Volt) Oxymesterone 534>389 534>444 (-30Volt) (-25Volt) Chloromethylboldenone 478>240 478>373 (-20Volt) (-25Volt) Oxandrolone 378>321 378>363 (-10Volt) (-10Volt) 16-hydroxyfurazabol 490>143 490>231 (-40Volt) (-25Volt)

3 Safety and environment

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Printdatum: 1-Oct-08 r All samples and materials are discarded as described in SOP ARO/487.

4 Chemicals and reagents 4.1 Chemicals 4.1.1 MilliQ water is used throughout this procedure unless stated otherwise. 4.1.2 Sodium acetate (Merck, art. 6268). 4.1.3 Acetic acid (Merck, art. 63). 4.1.4 Acetate buffer 2 mol/l, pH 5.2. Dissolve 25.2 g acetic acid and 129.5 g sodium acetate in water, add water to a final volume of 1000 ml and adjust the pH. 4.1.5 Suc d'Helix Pomatia containing 100.000 units β-glucuronidase and 1000.000 units sulfatase per ml. (Industrie Biologique, France, code IBR 213473). 4.1.6 Tris-(hydroxymethyl)-aminomethane (Merck, art. 8382). 4.1.7 Tris buffer, 0.1 mol/l, pH 9.5. Dissolve 12.1 g Tris in water. Adjust the pH at 9.5 and add water to a final volume of 1000 ml.

4.1.8 SPE extraction column: 3 ml disposable C18 cartridge (Varian, art. 1210-2052). 4.1.9 SPE extraction column: disposable Oasis cartridge (Waters, art. WAT094226). 4.1.10 Methanol (Biosolve, art. 13683502). 4.1.11 Acetone (Biosolve, art. 01030501). 4.1.12 Iso-octane (Merck, art. 104718). 4.1.13 TertitaireButylMethylEther (TBME) (Biosolve, art. 13890602). 4.1.14 Derivatisation reagent: MSTFA (Alltech, art. 18038). 4.1.15 Ammonium iodide (Fluka, art. 09874). 4.1.16 Dithiothreitol (BDH, art. 104582J). 4.1.17 Derivatisation mixture: MSTFA ++: dissolve 12,8 mg dithiothreitol and 6,4 mg ammoniumjodide in 4 ml MSTFA. 4.2 Standards Stock solutions containing 1 mg/ml are prepared by dissolving the appropriate amount of the analyte in ethanol. The solutions are stored in the dark at -20°C for a maximum period of 5 years. Working solutions are prepared by 10-fold dilutions of the stock solution with ethanol. These solutions are stored in the dark at 4°C (range 1-10°C) for a maximum period of 12 months. Internal Standard Relevant standard estradiol-d3. As internal standard estradiol-d3 is attached, the amount is 50 µl from 0,1 ng/µl (5 ng). Standards Stock solutions containing 1 mg/ml and working solutions of 0,1 mg/ml are prepared. A standard mixture is made by pipetting 25 µl from each solution of 0,1 mg/ml in a total amount of 25 ml ethanol. This standard mixture contains 0,1 ng/µl ethanol. Relevant data of the analytes are listed in Table 2. Table 2. Information analytes. Analyte CAS Formula Mol. Weight (g/mol)

Dromostanolone 58-19-5 C20H32O2 304.5

Methenolone 153-00-4 C20H30O2 302.5

Mibolerone 3704-09-4 C20H30O2 302.5

Calusterone 17021-26-0 C21H32O2 316.5

Oxymesterone 145-12-0 C20H30O3 318.5

Chloromethylboldenone n.a. C20H27ClO2 334.2

Oxandrolone 53-39-4 C19H30O3 306.5

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Printdatum: 1-Oct-08 r 16-hydroxy- 36455-74-0 C20H30N2O3 346.5

Estradiol-d3 (internal standard) n.a. C18H21D3O2 275.2 4.3 Materials 4.3.1 Glass tubes (10 ml) (Omnilabo, art. 206029). 4.3.2 Automatic pipettes (Gilson P100, P200, P1000 and P5000). 4.3.3 pH indicator strips pH 4.0-7.0 (Merck, art. 9542). 4.3.4 Vortex (Vibrofix, FV1). 4.3.5 Vacuum manifold (Varian, VAC-ELUT 20) . 4.3.6 Electric waterbath with thermostat adjustable ± 5 °C with nitrogen facility (TurboVap, Zymark). 4.3.7 Varifuge 3.0R, centrifuge (Heraeus). 4.3.8 Glass derivatisation vials (Alltech, art. 3112829) with cap (Alltech, art.73044) and insert (Alltech, art. 73060). 4.3.9 Injection vials, (Agilent, art. 5182-0714), with 50 µl inserts (Alltech, art. 98024) and caps (Agilent, art.5182-0717). 4.3.10 GC-MSMS equipment. Varian 1200L. 4.3.11 Fused silica capillary GC-column VF-17MS, length 30 meter, i.d. 0.25 mm, 0.25 micron film thickness (Varian, art. CP8982). 4.3.12 Incubator thermostat adjustable ± 3°C (Salvis). 4.3.13 Heating module thermostat adjustable ± 5°C (Pierce, art.18790) with nitrogen facility.

5 Procedure 5.1 Spiking procedure A portion of 5 ml of urine is transferred to a 10 ml glass tube. The samples are spiked with β-estradiol- d3 at the level of 5 ng (corresponding to 1 ng/ml). Also control samples are prepared. These control samples consist of the urine sample and the same urine sample spiked at 2 ng/ml, see table 3. Table 3. Pipetting scheme for samples and control samples. ID. Standard mixture Internal standard E2-d3 Sample 0 µl 50 µl of 0.1 ng/µl Sample spiked on 2 ng/ml 100 µl of 0.1 ng/µl 50 µl of 0.1 ng/µl To the samples is 2 ml acetate buffer added and 25 µl helix pomatia, check the pH and adjust if necessary. Hydrolysis is performed overnight at 37°C. 5.2 Solid Phase Extraction C18 A SPE C18 column is preconditioned with 5 ml of methanol and 5 ml of water. The sample (5.1) is passed through the column. The C18 column is washed with 5 ml of water and 4 ml of 40/60v/v-% methanol/water. Compounds are eluted with 4 ml 80/20 v/v-% methanol/water followed by 2 ml of methanol. The eluate is evaporated at 55°C under a gently stream of nitrogen until the volume is smaller then 0.5 ml. 5.3 LLE The extract is dissolved in 2 ml of tris buffer (pH 9.5). The buffer is extracted twice with 4 ml of TBME. The organic extracts are combined and evaporated at 55°C under a gently stream of nitrogen until dryness. The extract is reconstituted in 0.3 ml methanol and 2.7 ml water. 5.4 Solid Phase Extraction OASIS A SPE oasis column is preconditioned with 3 ml of methanol and 3 ml of water. The sample (5.3) is passed through the column. The column is washed with 3 ml of water and 3 ml of 40/60v/v-%

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Printdatum: 1-Oct-08 r methanol/water. Compounds are eluted with 4 ml 80/20 v/v-% methanol/water followed by 2 ml of acetone. The eluate is evaporated at 55°C under a gently stream of nitrogen until dryness. The dried extract is reconstituted in 300 µl ethanol. 5.5 Detection A calibration curve is prepared by pipetting the volumes given in table 4 into derivatisation vials. Table 4. Preparation of calibration curve ID. Standard mixture Internal standard Blank 0 µl 50 µl of 0.1 ng/µl St 10 ng 100 µl of 0.1 ng/µl 50 µl of 0.1 ng/µl St 7.5 ng 75 µl of 0.1 ng/µl 50 µl of 0.1 ng/µl St 5.0 ng 50 µl of 0.1 ng/µl 50 µl of 0.1 ng/µl St 2.5 ng 25 µl of 0.1 ng/µl 50 µl of 0.1 ng/µl St 1.25 ng 12.5 µl of 0.1 ng/µl 50 µl of 0.1 ng/µl

The extracts and standards are transferred into derivatisation-vials and evaporated at 60 °C under a gentle stream of nitrogen until dryness. The dry residue is derivatised by adding 25 µl MSTFA++ followed by incubation of 1 hour at 60°C. The derivatised mixture is evaporated at 60°C under nitrogen until dryness. The extract is reconstituted in 50 µl iso-octane and transferred into an injection-vial with micro insert. The vials are placed in the automatic sampler and 2 μl is injected into the GC-MSMS, data acquisition is performed as described in table 2.

6 Calculation 6.1 Performance check The first step is a performance check of the GC-MSMS system after tuning (full autotune). This is done by injecting a 20 pg standard estradiol-d3-di-TMS (absolute injection) and determination of the S/N ratio. The S/N-ratio at MRM 419>285 should be greater then 200. The same standard is injected in SIM mode, the S/N ratio of m/z 419 should be greater then 500. 6.1 Calculation The peak-area of the selected of the standard and internal standard are divided and the calculated ratio is the response variable. A calibration curve is constructed by linear curve fitting using linear regression calculation. Unknown concentrations are calculated by interpolation.

7 Validation and Measurement uncertainty Since there were no relevant isotope labeled internal standards were available the method can not be categorized as a quantitative method, and is therefore a qualitative screening method. According to the 2002/657/EC the following validation parameters have to be determined: Selectivity/specificity and the Detection capability (CCß).

Validation was performed by analysing 95 samples. The samples were also spiked at 2 ng/ml. The selectivity/specificity is demonstrated by the analysis of the blank samples. There should be no signal present at the expected retention time of the analyt. All samples were from different animals. By doing so the matrix effect and indirect the ruggedness of the method is determined. The validation was performed on different days.

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Printdatum: 1-Oct-08 r To determine the CCß in at least 95% of the spiked samples the analyte of interest should be detected (≤ 5% false compliant results).

In the table below an overview of the validation result is given

Table 5. Overview of the validation results (n=95 blank, and the same 95 samples spiked at 2 ng/ml)

Compound Blank samples Spiked samples dromostanolone 95 samples compliant 95 samples non-compliant (100%) methenolone 95 samples compliant 92 samples non-compliant (97%) mibolerone 95 samples compliant 83 samples non-compliant (87%) calusterone 95 samples compliant 90 samples non-compliant (95%) oxymesterone 95 samples compliant 95 samples non-compliant (100%) chloromethylboldenone 95 samples compliant 94 samples non-compliant (99%) oxandrolone 95 samples compliant 54 samples non-compliant (59%) 16-hydroxyfurazabol 95 samples compliant 95 samples non-compliant (100%)

The percentage of false compliant results for mibolerone and oxandrolone is larger then five percent. This indicates that the CCß for these compounds is larger ten 2 ng/ml.

8 Quality control Quantification is only valid if: • The maximum of the signal originating from the analyte detected with a S/N ratio > 3. • In the spiked control samples the S/N ratio of the signal should be > 6. • The coefficient of correlation of the constructed calibration curve is better than 0.985.

10 Relating documents SOP ARO/475, Method validation using ResVal, revision 2, 21april 2004, RIVM. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results

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Printdatum: 1-Oct-08 r Documentbeheer Algemeen Invoerdatum: 20 maart 2007 Wijzigingsdatum: 24 april 2007 Controledatum: 22 april 2012 Publicatiedatum: 24 april 2007 Wijzigingen ten opzichte van vorige versie:

Beoordelaars Marco Blokland (Onderzoeker) Leen van Ginkel (labhoofd) Saskia Sterk (afdelingshoofd)

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