Screening and confirmation of sulfonamides, trimethoprim and dapsone in animal tissues using HPLC-DAD

Austrian Agency for Health and Food Safety (AGES) Screening and confirmation of sulfonamides, trimethoprim and dapsone in animal tissues using HPLC-DAD Prepared by Checked by QM-Controller Approved by Name Regina Schuh Martin Brandtner Irina Schwaiger-Nemirova Thomas Kuhn Date 02.12.2010 02.12.2010 02.12.2010 02.12.2010 Signature signed signed signed signed QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 1

1. PREVIOUS VERSION 1.1. Replaced document First version therefore not applicable 1.2. Changes to previous version Not applicable 2. AREA/FIELD/SCOPE OF APPLICATION 2.1. Aim/Purpose/Object Sulfonamides are antibacterial drugs, the maximum residue levels (MRL s) in table 1 of the regulation (EU) Nr. 37/2010 are regulated as the cumulative value, i.e. the residues of all substances of the sulfonamide group should not exceed a total of 100 µg/kg. Trimethoprim is no sulfonamide, but is often used in combination with these (MRL: 50µg/kg; equidae: 100 µg/kg). Dapsone is similar to sulfonamides cocerning chemical structure but strictly speaking, none. Dapsone is listed in table 2 of the regulation (EU) Nr. 37/2010. Its application is prohibited. The method is used to monitor the listed substances in animal tissues with respect to their presence and their maximum residue levels (MRL s) respectively. 2.2. Investigated parameters CAS: Dapsone (4,4'-diamino-diphenyl sulfone) [80-08-0] Sulfabenzamide [127-71-9] Sulfacetamid [144-80-9] Sulfachlorpyrazin (Sulfaclozin) [102-65-8] Sulfachlorpyridazin [80-32-0] Sulfadiazine (Sulfapyrimidin) [68-35-9] Sulfadimethoxin [122-11-2] Sulphadimidine (sulfamethazine) [57-68-1] Sulfadoxine [2447-57-6] Sulfamerazine [127-79-7] Sulfameter (Sulfametoxydiazin) [651-06-9] Sulfamethizole [144-82-1] Sulfamethoxazole [723-46-6] Sulfamethoxypyridazin [80-35-3] Sulfamonomethoxin [1220-83-3] Sulfamoxol [729-99-7] Sulfaphenazol [526-08-9] Sulfapyridine [144-83-2] Sulfaquinoxaline [59-40-5] Sulfathiazole [72-14-0] Sulfatroxazol [23256-23-7] Sulfisomidine (internal standard) [515-64-0] QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 2

Sulfisoxazol (WHO: Sulfafurazol) [127-69-5] Trimethoprim [738-70-5] 2.3. Sample types Animal tissues (muscle, liver, kidney, fish) 3. METHOD 3.1. Principle The amino groups in the molecular structures of the analytes make a solid phase extraction (SPE) using cation-columns possible. After the chromatographic separation with HPLC, the analytes are detected by UV absorption. UV spectra and retention times are used for identification of substances. 3.2. Summary Samples are homogenized in acetonitrile, then defatted with n-hexane. Further purification is carried out with solid phase extraction (cation exchange columns; OASIS MCX). Samples are measured by high performance liquid chromatography (HPLC) coupled with diode array detection (DAD). 4. USED TERMS, ABBREVIATIONS AND SYMBOLS rpm: rotations per minute RT: room temperature SPE: solid phase extraction 5. WARN- AND SAFETY ADVICES Organic solvents and chemicals have to be seen as potential hazards. Direct contact with skin, exposure to vapour should be avoided. Methanol toxic highly flammable Acetonitrile harmful to health highly flammable n-hexane harmful to health highly flammable environmental hazard Care should be taken in the case of handling with acids and bases. ph adjustment has to be done slowly. Hydrochloric acid corrosive Please see safety data sheets! Acetic acid corrosive Please see safety data sheets! QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 3

Ammonia corrosive Please see safety data sheets! 6. EQUIPMENT/FACILITIES AND REQUIRED MATERIALS The mentioned company names are used to indicate type and quality of products. Other products can also be used if they meet the requirements. In addition to the usual laboratory equipment you need the following equipment/tools: 6.1. Devices - Moulinette kitchen blender (e.g. Moulinex) - Balance (e.g. Mettler PM 4000) - Centrifuge (e.g Heraeus Cryofuge 6000i, rotor 6606) - Nitrogen-evaporator (e.g. Pierce Reacti-Vap III) - ph-meter (e.g. Orion 420 A) - Shaker (e.g. VWR Mini Vortex mixer) - Piston - SPE-vacuum chamber (e.g. J. T. Baker) - Vacuum pump + controller (e.g. Vakumbrand) - Homogenizer (e.g. Janke & Kunkel Ultra Turrax) - Ultrasonic bath (e.g. Bandelin Stonorex Super RK510) - HPLC with DAD (e.g. Waters Alliance 2690, PDA 2996) 6.2. Required materials Pleated filter Sterile filter (0.2 microns) SPE columns OASIS MCX (6 ml, 500 mg) 50 ml Greiner tubes 10 ml Greiner tubes 1-ml syringes Injection needles d = 0.9 x 50 mm HPLC-Vial (PP 400 µl) Funnel Volumetric flask (50, 100, 1000ml) Combi-Tip 7. REAGENTS, SOLUTIONS AND TESTORGANISMS 7.1. Reference standards - Dapsone (4,4'-diamino-diphenyl sulfone) Sigma D-2505 - Sulfabenzamide Sigma S-9757 - Sulfacetamid Sigma S-8627 - Sulfachlorpyrazin (Sulfaclozin) Chemos - Sulfachlorpyridazin Sigma S-9882 - Sulfadiazine (Sulfapyrimidin) Sigma S-8626 - Sulfadimethoxin Sigma S-7385 QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 4

- Sulphadimidine (sulfamethazine) Sigma S-6256 - Sulfadoxine Riedel de Haen 46 810 - Sulfamerazine Sigma S-8876 - Sulfameter (Sulfametoxydiazin) Sigma S-0383 - Sulfamethizole Sigma S-5632 - Sulfamethoxazole Sigma S-7507 - Sulfamethoxypyridazin Sigma S-7257 - Sulfamonomethoxin Sigma S-0508 - Sulfamoxol Sigma D-6549 - Sulfaphenazol Pfizer - Sulfapyridine Sigma S-6252 - Sulfaquinoxaline Sigma S-7382 - Sulfathiazole Sigma S-0127 - Sulfatroxazol Leo Pharm - Sulfisomidine Riedel de Haen 46 908 - Sulfisoxazol (WHO: Sulfafurazol) Sigma S-6377 - Trimethoprim Sigma T-7883 7.2. Chemicals / Reagents The mentioned company names are used as an indication of the quality of the products. Products of other manufacturers can also be used if they meet the requirements. - Acetonitrile for HPLC Merck 14 291 - Acetic acid (100%) Merck 1.00063 - Ammonia Merck 1.05432 - n-hexane p. a. Merck 1.04367 - Methanol LiChrosolv Merck 1.06018 - Ammonium acetate p. a. Merck 1.01116 - Hydrochloric acid 37% Merck 1.00317 7.3. Testorganisms Not applicable 7.4. Gases Nitrogen (from the central gas supply) 7.5. Solutions / culture media Unless otherwise indicated, an aliquot or a multiple of the given solutions can be produced with the required accuracy. 7.5.1. 20 mm ammonium acetate buffer (ph 4.0) Weigh 1.54 g ammonium acetate in a 1000 ml beaker and dissolve with 1000 ml ultrapure water. Adjust with concentrated acetic acid (about 6 ml) at ph 4.0, and filter through a 0.45µ filter. 7.5.2. Stock standard solutions (1 mg/ml) QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 5

Dissolve 10 mg of substance in a 10 ml volumetric flask with methanol and fill up to the mark (ecxeption: only 1 mg of sulfadiazine [0.1 mg/ml], due to the lower solubility in methanol). The solutions are stable at -20 C for at least 5 years. 7.5.3. Internal standard (10 µg/ml) Put 1 ml sulfisomidine stock standard solution (6.5.2.) in a 100 ml volumetric flask and fill with ultrapure water to 100 ml (final conc.: 10 µg/ml). If required, an other sulfonamide can be used as internal standard. 7.5.4. Spiking standard "SUL 1X" Put 200 µl of sulfacetamide, sulfadiazine, sulfathiazole, sulfemerazin, sulfamoxol, sulfemethizole, sulfadimidine, sulfameter, sulfamonomethoxine, sulfachlorpyridazine, sulfamethoxazole, sulfabenzamide, sulfadimethoxin, sulfachlorpyrazin, sulfaquinoxalin, sulfaphenazol, 60 µl of dapsone, and 100 µl of trimethoprim stock standard solutions (6.5.2.) in a 20 ml volumetric flask and fill with ultrapure water to 20 ml (final conc.: 10 µg/ml for sulfonamides, 3 µg/ml dapsone, 5 µg/ml trimethoprim). 7.5.5. Spiking standard "SUL 2X" Put 50 µl of sulfadoxine, sulfamethoxypyridazin, sulfapyridine stock standard solutions (6.5.2.) in a 5 ml volumetric flask and fill with ultrapure water to 5 ml (final conc.: 10 µg/ml). 7.5.6. Spiking standard "SUL 3X" Put 50 µl of the required sulfonamide stock standard solution (6.5.2.) in a 5 ml volumetric flask and fill with ultrapure water to 5 ml (final conc.: 10 µg/ml). 7.5.7. 1 M hydrochloric acid Put 100 ml 37% hydrochloric acid in a 1000 ml flask (filled with about 200ml ultrapure water) and fill with ultrapure water to 1000 ml. 7.5.8. 0.1 M hydrochloric acid Put 10 ml 37% hydrochloric acid in a 1000ml flask (filled with about 200ml ultrapure water) and fill with ultrapure water to 1000 ml. 7.5.9. Eluent: ammonia in acetonitrile (1.375%) Put 60 ml acetonitrile in a 100 ml flask, pipet 5.5 ml conc. ammonia (25%) into it and fill with acetonitrile to 100 ml. The solution is freshly prepared before each analysis. 7.5.10. Saturated sodium sulfate solution Dissolve appr. 300g sodium sulfate in 1 l pure water until a sediment remains visible. 7.5.11. Calibration curves Calibration curves are established within the linear range, e.g.: Sulfonamides: 0.625 µg/ml - 10 µg/ml Trimethoprim: 0.3125 µg/ml - 5 µg/ml Dapsone: 0.1875 µg/ml - 3 µg/ml 7.6. Disposal QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 6

Organic solvents are disposed in the waste canisters for solvents located in the laboratories. The further disposal plan is defined separately. 8. SAMPLING, SAMPLEPREPARATION AND STORAGE Preparation of samples: connective tissue and fat are removed, samples homogenized (minced); weigh 10.0 g with an accuracy of ± 0.1 g into the test tube and store it until further analysis at -20 C. 9. PROCESSING 9.1. Prearrangements 9.1.1. Preparation of control samples In order to determine the recovery rate at least one negative control sample (10.0 g) of the respective matrix material is fortified with 100 µl spiking standard SUL 1X (6.5.4.). The further processing is carried out in parallel with the samples to be tested. Note: If you have to analyse a sulfonamide, which spiking standard SUL 1X (6.5.4.) does not contain, use the corresponding spiking standard SUL 2X or 3X (6.5.5. or 6.5.6.). 9.2. Sample Processing 1. Extraction Add 100 µl internal standard (6.5.3.) to all samples and control samples and wait for at least 10 minutes Add 10 ml acetonitrile and shake at least 10 minutes Add 10 ml saturated sodium sulfate solution (6.5.10.) and shake at least 10 minutes Centrifuge 10 minutes at 3600 g Transfer upper phase (acetonitrile) in a 50ml tube Add 10 ml pure water, 10 ml 1M HCl (6.5.7.) and 10 ml n-hexane and shake at least 10 minutes Centrifuge 10 minutes at 3000 g Remove supernatant (n-hexane), fill up to 50 ml with pure water, close the tube and shake 9.2.2. Solid Phase Extraction (SPE) OASIS MCX SPE columns (6 ml, 500 mg): Condition SPE columns with 5 ml methanol, and then 5 ml of pure water without vacuum Transfer sample extract on SPE columns without vacuum Rinse sample tubes with 5 ml of 0.1 M hydrochloric acid (6.5.8.) and transfer it on SPE columns, then add directly 5 ml of 0.1 M hydrochloric acid (6.5.8.) on SPE columns, then add directly 5 ml methanol on SPE columns without vacuum Run 10 minutes dry (600mbar) Elute with 5 ml elution solution (6.5.9.) without vacuum Run dry shortly (600mbar) Evaporate the eluate under nitrogen at 40 C to dryness QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 7

Dissolve in 200 µl pure water/acetonitrile (19+1 v / v), shake (vortex) and 5 min ultrasonic bath; if solution is turbid, centrifuge 15 min at 13000g Pipette 100 µl of used spiking standard SUL 1X or 2X or 3X (6.5.4. or 6.5.5. or 6.5.6.) and 100 µl of internal standard (6.5.3.) into a tube, mix, transfer into a vial and seal. 9.2.3. Measurement / analysis The qualitative and quantitative analysis is carried out by HPLC and diode array detector (DAD), for example under the following conditions (more detailed information, see method description of software): HPLC column: Synergi Polar RP; 250 mm x 2 mm, 4 µm Guard column: Phenomenex Security Guard Polar RP; 4mm x 2mm Mobile phase: A: 0.01 M ammonium acetate buffer (ph 4.0) (6.5.1.); B: acetonitrile Gradient: 0 min: 95% A, 16 min: 60% B, 17 min: 95% A (20 minutes) Flow: 0.35 ml/min Detection: UV: 265nm, 292.6 nm HPLC column Temperature: 40 C Injection volume: 50 µl 10. EVALUATION 10.1.Identification / Calculation 1. Identification Sulfonamides, dapsone and trimethoprim are identified by: comparing the relative retention times with those of positive control sample(s) analyzed in the same sequence. For identification relative retention times must match with a tolerance of ± 2.5%. comparing the UV spectrum of the analyte with the corresponding UV spectrum of the spectral library and/or the positive control sample(s) respectively. 2. Calculation The extract from 10 g sample is analyzed in 200 µl test solution. Assuming a sample concentration of 100 µg/kg (1 µg in 10 g sample), results in a hypothetical concentration (assuming 100% recovery) in the solution of 1 µg/ 200 µl = 5 µg/ml. This gives the following formula: 5 g/ml in the test solution corresponds to 100 µg/kg (ppb) in the sample All results must be corrected by recovery. When an analyte is detected, the corresponding sample is usually analysed twice again. Dapsone is a forbidden substance (table 2 of the regulation (EU) Nr. 37/2010) and must be confirmed by a mass spectroscopic method (2002/657/EC). 10.2.Protocol QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 8

The protocol includes at least: name(s) or signature(s) of the analyst(s) (sample preparation and measurement) dates of sample preparation and measurements list of used standards and control samples and the samples tested (with clearly identifiable sample names or numbers) chromatograms of the samples and control samples with peak areas and concentrations results of samples if applicable deviations from the SOP 10.3.Results Concentration of an analyte above the quantitation limit (LOQ): result in µg/kg Concentration of an analyte between its detection limit (LOD) and its quantitation limit (LOQ): detectable (< LOQ) Concentration of an analyte below the detection limit (LOD): not detectable (LOD and LOQ are given in µg/kg) 11. VALIDATION The results of the validation are summarized in the accompanying validation folder or electronic file (location: room or electronic path). 12. LITERATURE 12.1.Scientific Literature W. Hela, M. Brandtner, R. Widek, R. Schuh: Food Chemistry 83 (2003), 601-608 12.2.Rules, Legislation, Law, Guidelines Regulation (EU) No 37/2010 of 22. December 2009 (MRL s in the EU) Commission Decision (EU) of 12. August 2002 (2002/657/EC) implementing the Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results Location: room or electronic path QM-Coding: PV_CC_VIE_TAHO_105_06 Status: Valid as of: December 6, 2010 Page! 9