R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 1 van 10 the Environment Revision : 0 Date : 2005.01.13 1. INTRODUCTION

This method describes the analysis of thyreostatics, thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU), tapazol (TAP) and mercaptobenzimidazol (MBI), in samples bovine urine. The clean up consist of hydrolysis of the samples, liquid-liquid extraction (LLE) with acetonitril/dichloromethane, derivatisation with 7-chloro-4-nitrofurazan (NBD-chloride) and liquid chromatography in combination with mass spectrometric detection (LC-MS).

2. 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 RIVM to the exclusion of the others which might also be suitable.

2.1 Chemicals and reagents

All chemicals including standards and solutions are of defined quality. Pure chemicals are of “Pro Analyse” quality or better, used water is of bidest or better quality.

2.1.1 Tapazol, Sigma-Aldrich Chemie. 2.1.2 Thiouracil, Sigma-Aldrich Chemie 2.1.3 Methylthiouracil, Sigma-Aldrich Chemie. 2.1.4 Propylthiouracil, Sigma-Aldrich Chemie 2.1.5 Dimethylthiouracil, Sigma-Aldrich Chemie 2.1.6 Mercaptobenzimidazol, Sigma-Aldrich Chemie. 2.1.7 Acetonitril, Biosolve. 2.1.8 Dichloromethane, Merck. 2.1.9 Ethylacetate, Merck. 2.1.10 Acetic acid, J.T. Baker. 2.1.11 Methanol, Biosolve. 2.1.12 Ethanol, Biosolve. 2.1.13 Sodium chloride, Sigma-Aldrich Chemie. 2.1.14 Sodium sulphate anhydrous, BDH Laboratory Supplies. 2.1.15 Potassium dihydrogen phosphate, J.T. Baker. 2.1.16 Sodium monohydrogen phosphate, J.T. Baker. 2.1.17 7-Chloro-4-nitrofurazan, Sigma-Aldrich Chemie. 2.1.18 Acetonitril/dichloromethane (75/25; v/v). Mix 300 ml of acetonitril with 100 ml

dichloromethane. 2.1.19 2 Mol/l phosphate buffer pH 8.0. Dissolve 1.50 g potassium dihydrogen phosphate and

26.8 g sodium monohydrogen phosphate in 1000 ml of water. The pH is adjusted with acetic acid to 8.0 ± 0.1.

2.1.20 Solvent A: methanol/5 mmol ammonium acetate (10/90; v/v). Dissolve 0.35 g ammonium acetate in 900 ml of water ad 100 ml of methanol.

2.1.21 Solvent B: methanol/water (90/10 v/v). Mix 100ml of water with 900 ml of methanol. 2.1.22 Solvent D: methanol/ethanol (50/50 v/v). Mix 500 ml of methanol with 500 ml ethanol. 2.1.23 Methanol/water (40/60 v/v). Mix 40 ml of water with 60 ml of methanol.

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 2 van 10 the Environment Revision : 0 Date : 2005.01.13 2.1.24 Standards for PTU, TAP or MBI. Weight 5mg of PTU, TAP or MBI and add 5ml of

methanol (1 mg/ml). 2.1.25 Standards for TU or MTU. Weight 5mg of TU or MTU and add 50ml of methanol

(0.1 mg/ml or 100 ng/ml).

2.2 Apparatus

Standard laboratory glassware and equipment is used, with addition of: 2.2.1 Glass tubes 10 ml. 2.2.2 pH-meter, Schott. 2.2.3 Heating module thermostat adjustable ± 50C with nitrogen facility. 2.2.4 HPLC-vials (2ml). 2.2.5 Centrifuge, Varifuge 3.0R with rotor 5315, Hereaus. 2.2.6 Oven, Memmert. 2.2.7 Column, lichrocart 125-2 hplc superspher 100RP-18, 4 um, end capped, Merck. 2.2.8 The LC-MS system consists of a Finnigan LCQ Deca-system and Alliance LC-pump

and autosampler. 3. ANALYTICAL PROCEDURE A gradient system is used (see table 1). Table 1 LC- gradient.

Time (minutes)

Flow ml/min

Solvent A (%)

Solvent B (%)

Solvent D (%)

0.00 0.25 70 30 0 2.00 0.25 70 30 0 13.00 0.25 10 90 0 14.00 0.25 10 90 0 14.01 0.25 0 0 100 16.00 0.25 0 0 100 16.01 0.25 70 30 0 21.00 0.25 70 30 0

The LCQ Deca is used in the MS2 mode, using the ESI(+) ionisation. Acquisition

parameters were vaporiser 5000C; capillary temperature 3500C; nitrogen (high purity). Table 2 Screening and confirmatory ions of the thyreostatics

Analyte Pseudo Molecular ion [M+H]+

Transition ions, used for screening

Transition ions, used for confirmation

Dimethylthiouracil 320 257 274 Mercaptobenzimidazol 314 238 268 Methylthiouracil 306 243 260 Propylthiouracil 334 271 288 Tapazol 278 232 248 Thiouracil 292 229 246

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 3 van 10 the Environment Revision : 0 Date : 2005.01.13 3.1 Urine sample preparation

Samples of urine are stored in the dark at -200C or at +40C if analysis is foreseen to be within two days.

3.1.1 Homogenise the sample urine. 3.1.2 Pipette 1ml of urine in a test tube of 10 ml. 3.1.3 Pipette 50µl of 1 ng/µl DMTU. 3.1.4 Add 200 mg sodium chloride. 3.1.5 Vortex for 30 seconds. 3.1.6 Add 4 ml of acetonitril/dichloromethane (75/25; v/v). 3.1.7 Vortex for 30 seconds. 3.1.8 Centrifuge for 3 min. 3600 rpm or 2800 g. 3.1.9 Transfer the top layer to a new test tube. 3.1.10 Add 8 ml of acetonitril/dichloromethane (75/25; v/v). 3.1.11 Vortex for 30 seconds. 3.1.12 Centrifuge for 3 min 3600 rpm or 2800 g 3.1.13 Transfer the top layer to the new test-tube. 3.1.14 Evaporate the collected top layer under a stream of nitrogen at 500C and repeat the

procedure 3.1.10 until 3.1.13. 3.1.15 Evaporate the collected top layers to dryness under a stream of nitrogen at 500C. 3.1.16 Add 5 ml of phosphate buffer pH 8.0. 3.1.17 Freshly made NBD-chloride solution; weight 5 mg of NBD-chloride and add 2 ml of

methanol, vortex for 30 seconds. 3.1.18 Add 0.1 ml of the fresh NBD-Chloride solution. 3.1.19 Derivatise for 1 hour at 400C. 3.1.20 Cool the sample to room temperature. 3.1.21 Add 5 ml of ethyl acetate and vortex for 30 seconds. 3.1.22 Centrifuge for 3 min 3600 rpm or 2800 g. 3.1.23 Transfer the top layer to a new test tube. 3.1.24 Evaporate the top layer under a stream of nitrogen at 500C till 3 ml. 3.1.25 Repeat twice the procedure 3.1.20 until 3.1.24 (collect the top layers in the same test tube). 3.1.26 Evaporate the collected top layers to dryness under a stream of nitrogen at 500C. 3.1.27 Add 125 µl of methanol/water (40/60; v/v) and vortex for 30 seconds. 3.1.28 Transfer the solvent to a HPLC-vial with insert. 3.1.29 Inject 100 µl into the LCQ-Deca. 3.2 LC-MS2 -screening analysis

From the standards solutions of MBI, MTU, PTU, TAP and TU amounts of 0, 25, 50, 100, 150 and 250 ng/125 µl methanol/water (40/60; v/v) per standard and from DMTU 50 ng/125µl methanol/water (40/60; v/v) are made.

For screening purposes the extract of the unknown samples of urine and liver, together

with the blank sample and at least two fortified samples (25 ng/µl and 50 ng/µl= control urine) are made.

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 4 van 10 the Environment Revision : 0 Date : 2005.01.13 3.3 Quantification Quantitative results are obtained by constructing a calibration curve based on the linear

regression of the ratio analyte/DMTU versus the concentration. For this purpose the software program CALWER and RESVAL used (5.1).

The detector response is the response obtained for the second transition ion (see table 3 ’Screening ions’). Concentrations of thyreostatics in standards depend on the concentration found in the suspected sample in such a way that the response for the unknown sample for the unknown sample is between the responses obtained for the calibrants. Together with the standards for the calibrations of the specific thyreostatics is calculated using the CALWER software program.

Quantification is only valid if: - The maximum of the signal originated form the analyte in the suspected sample has a

S/N ratio ≥ 3. - The coefficient of the correlation of the constructed curve is > 0.96.

3.4 LC-MS2 confirmation analysis The results of the analysis can only be stated “non compliant” if the presence of the

analyte in the sample is confirmed according to the criteria specified for LC-MSn laid down in the 2002/657/EC (2). For MBI, MTU, PTU, TAP, TU measurement by LC-MS2, MS2-ions have to be monitored and the ratio between the two recorded fragment ions has to be calculated and compared with the ratio as obtained for either standards or fortified control samples. Table 2 shows the specific ions for the thyreostatics. For confirmation of the identity of the thyreostatics, the sample of urine is re-analysed sample(s) a blank sample of urine fortified at a level corresponding with the unknown analyte of interest is analysed. Alternatively the confirmatory analysis can be combined with the analysis of quantification.

4. VALIDATION OF METHOD

The method described in this SOP was validated conform ARO/475. The method should be capable of detecting thiouracil methylthiouracil, propylthiouracil, tapazol and mercaptobenzimidazol at 50ng/ml urine, this is also the validation level used for this method. See Annex 1. In table 3 and 4 a summary of the validation result is shown.

Table 3 CCα and CCβ

Analyte in urine: CCα (ng/g) CCβ (ng/g) Measurement

uncertainty (U) Mercaptobenzimidazol (n=59) 9.46 16.12 22.15 Methylthiouracil (n=59) 5.77 9.83 56.42 Propylthiouracil (n=60) 8.44 13.78 26.01 Tapazol (n=60) 8.09 4.38 43.13 Thiouracil (n=60) 12.06 20.55 46.43

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 5 van 10 the Environment Revision : 0 Date : 2005.01.13 5. RELATED DOCUMENTS 5.1 Revision of Commission Decision 96/23/EC (2002). Implement Council Directive

96/23/EC concerning the performance of analytical methods and the interpretation of results.

5.2 Resval V2.0 Validation Report (SOP ARO/475). 5.3 K. De Wasch, H.F. de Brabander, S. Impens, M. Vandenwiele, D. Courtheyn,

Determination of mercaptobenzimidazol and other thyreostats residues in thyroid tissue and meat using high-performance liquid chromatography mass-spectrometry. J. of Chromatogr A. 912 (2001) page 311-317.

5.4 S. Sterk, C. van de Kamp, L. van Ginkel, R. Stephany, Thyreostatics in farm Animals,

Regulatory residue analysis within the European Union proceedings workshop CRL970407-970409. CRL document 389002 071 January 1998 pages 61-62.

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 6 van 10 the Environment Revision : 0 Date : 2005.01.13 6 ANNEX VALIDATION REPORTS

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 7 van 10 the Environment Revision : 0 Date : 2005.01.13

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 8 van 10 the Environment Revision : 0 Date : 2005.01.13

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 9 van 10 the Environment Revision : 0 Date : 2005.01.13

R S.O.P. : ARO/440 National Institute of TITLE: Analysis of thyreostatics in Annex : 1 Public Health and bovine urine by LC-MS Page : 10 van 10 the Environment Revision : 0 Date : 2005.01.13