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Method for the Detection of Zeranol and SOP : ARO/442 Taleranol In

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Method for the Detection of Zeranol and SOP : ARO/442 Taleranol In TITLE: Method for the detection of zeranol and S.O.P. : ARO/442 taleranol in meat and liver Page : 1 of 9 . Revision : 0 Date : 990715 1. INTRODUCTION 1.1 Summary This SOP describes a screening and confirmation method for the determination of resorcyclic acid lactones (RAL’s) in meat and liver samples of bovine origin. A procedure is described for the detection of a-zearalanol (zeranol) and b-zearalanol (taleranol) by a combination of solid phase extraction (SPE) and GC-MS. The method consist of, digestion of the meat sample using the enzyme protease and hydrolysis of the liver sample using suc d’Helix Pomatia, extraction of the RAL’s with tert-butylmethylether (TBME), purification using reversed-phase C18 cartridges. This method can also be used for the metabolites a-zearalenol, b-zearalenol, zearalanone and zearalenone. When doing so validation for these compounds is necessary. 1.2 Field of application The method is used to perform routine screening in meat and liver samples and has proven to be suitable for the analysis of a-zearalanol and b-zearalanol. The limit of detection is 0.5 mg/g based on the detection of the most abundant diagnostic ions namely m/z 433 and m/z 437 for the internal standard, with a response at the correct retention time exceeding the signal to noise ratio of 3. The method is validated according to SOP ARO/425 (1.3.3). 1.3 References 1.3.1. H.J. van Rossum et al, Multi residue analysis anabolic agents, SOP ARO/113, revision 4, 21 January 1997, RIVM. 1.3.2. H.A. Herbold, S.S. Sterk, R.W. Stephany, L.A. van Ginkel. Multi residue method using coupled-column HPLC and GC-MS for the determination of anabolic agents in samples of urine. RIVM report n.389002 - 035, September 1997. 1.3.3. A.A.M. Stolker, Procedure for the validation of analytical methods, SOP ARO/425, revision 1, 28 April 1997, RIVM. 1.3.4. Report: European Commission Decision laying down requirements for analytical methods to be used for detecting certain substances and residue thereof in live animals and animal products according to Council Directive 93/256/EC. 1.3.5 S.M. Gort and R. Hoogerbrugge, Linear regression program Calwer. Report RIVM no. 502501025. A spreadsheet program for calibration. March 1995. 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 National Institute of Public Health and Environment (RIVM) to the exclusion of others which might also be suitable. TITLE: Method for the detection of zeranol and S.O.P. : ARO/442 taleranol in meat and liver Page : 2 of 9 . Revision : 0 Date : 990715 2.1 Chemicals 2.1.1 SPE extraction columns, 6 ml, 1 g, disposable octadecyl (C18) (Alltech, 205430). 2.1.2 SPE extraction columns, 6 ml, 1 g, disposable amino (NH2) (Alltech, 211153). 2.1.3 Methanol (Baker, 8045). 2.1.4 Ethanol (Baker, 8006). 2.1.5 Acetone (Baker, 8001). 2.1.6 Iso-octane (Baker, 8715). 2.1.7 Derivatization reagent: N,O-bis(trimethylsilyl)trifkuoracetamide (BSTFA) with 1% TMCS (Pierce, 38832). 2.1.8 Acetic acid (Merck, 63). 2.1.9 Sodium acetate (Merck, 6268). 2.1.10 Hydrochloric acid, 37% solution (Merck, 317). 2.1.11 Beta-glucuronidase/sulfatase (suc d'Helix Pomatia containing 100.000 units ß-glucuronidase and 100.000 units sulfatase per ml, France, code IBR 213473. 2.1.12 Acetate buffer, 2 mol/l, pH 5.2 Dissolve 25.2 g acetic acid and 129.5 g sodium acetate in 800 ml water. Adjust the pH to 5.2 ± 0.1, add water to a final volume of 1000 ml. 2.1.13 Tris buffer, 0.1 mol/l, pH 9.5. Dissolve 12.1 g of Tris(hydroxymethyl)-amino-methane (2.1.16) in 800 ml of water. Adjust the pH (5.5.18.) at 9.5 ± 0.1 and add water to a final volume of 1000 ml. 2.1.14 Tert-butylmethylether (TBME) (Merck, 1845). 2.1.15 Subtilisin (Protease) (Sigma, P-5380). 2.1.16 Tris(hydroxymethyl)-amino-methane (Merck, 8382) 2.2. Relevant Internal Standards 2.2.1 a-Zearalanol-d4 and b-zearalanol-d4 The analytes are a mixture of ± 50% of each compound. RIVM/ARO sample no. 87M1553. From these a standard stock solution containing 1 mg/ml is prepared. The stock solutions is prepared by dissolving the appropriate amount of the analytes in ethanol. Quality control includes the registration of a mass spectrum (identity). These solutions are stored in the dark at -20°C for a maximum period of 5 years. Working solutions are prepared by 10-fold dilution of the stock solutions with methanol. These solutions are stored in the dark at 4°C (range 1-10°C) for a maximum period of 6 months. 2.2.2 a-Zearalanol and b-zearalanol Relevant data of the analytes are listed in table 1. Stock solutions containing 1 mg/ml are prepared by dissolving the appropriate amount of the analytes in ethanol. Quality control includes the registration of a mass spectrum (identity). These solutions are stored in the dark at -20°C for a maximum period of 5 years. Working solutions are prepared by 10-fold dilution of the stock solutions with methanol. These solutions are stored in the dark at 4°C (range 1-10°C) for a maximum period of 6 months. Table 1. Data of analytes. Analytes CAS # Formula Mol. weight a-zearalanol 26538-44-3 C18H26O5 322.4 b-zearalanol 42422-68-4 C18H26O5 322.4 TITLE: Method for the detection of zeranol and S.O.P. : ARO/442 taleranol in meat and liver Page : 3 of 9 . Revision : 0 Date : 990715 2.3 Equipment 2.3.1 ASPEC® (Automated Sample Preparation with Disposable Extraction Columns) XL Gilson. The ASPEC® consists of three components: a model 401C dilutor, a sample processor and a set of racks and accessories to handle 6 ml SPE columns. The ASPEC® is programmed using the Gilson Sample Manager 721 software. 2.3.2 Collecting tubes for the ASPEC® system, Gilson type B54728-4. 2.3.3 Derivatization vials, Screw Top Vial /PTFE Septa (Omnilabo, 154920). 2.3.4 Automatic pipettes (Gilson P20, P100, P200, P1000 and P5000). 2.3.5 Injection vials, Wide Mouth Crimp (Alltech, 98213), with micro inserts (200 ml) (Alltech, EK-1022.395) and aluminium caps (Chrompack, 10210). 2.3.6 GC-MS equipment. Hewlett Packard 5890 series 11 gaschromatograph, equipped with a Hewlett Packard 7673 automatic sampler, a Hewlett Packard Vectra computer 486/66U with HPchem data acquisition software and a 5989A Mass Spectrometer (type Engine). 2.3.7 Fused silica capillary column CP SIL-5, Low bleeding/non polar, length 60 m, i.d. 0.25 mm, 0.1 micron film thickness, (Chrompack, 7818). 2.3.8 Vortex (Vortex-Genie). 2.3.9 pH-meter, (Applikon). 2.3.10 Moulinette S (Moulinex). 2.3.11 Electric water bath, thermostat adjustable ± 5°C, with nitrogen facility (TurboVap, Zymark). 2.3.12 Varifuge 3.0R, centrifuge with swing-out rotor 5315 (Heraeus). 2.3.13 Centrifuge tubes (50 ml), plastic (Omnilabo) 2.3.14 Incubator TH15, (Edmund Bühler). 3. GC-MS CONDITIONS Table 2: Parameters GC-MS Carrier gas helium 0.6 ml/min (EPC system for constant flow) Column 60 meter CP-SIL-5 Low Bleeding/non polar fused silica capillary (0.25 mm i.d. 0.1 mm film) Detector temperature 250oC MS-source, 120 o C quadruple. Oven temperature 80oC for 1 min, increased at 30oC/min. to 300oC and held at this temperature for 5 min. Ionisation EI - positive, SIM mode Injection volume 3 ml Pressure Electronic Pressure Control syst. Pressure is held at 50 psi for 1 minute, decreased at 99 psi/minute to 28.4 psi, constant during the oven-temperature program Mass monitored 433, 437 (internal standard) Tune High mass/sensitivity tune. TITLE: Method for the detection of zeranol and S.O.P. : ARO/442 taleranol in meat and liver Page : 4 of 9 . Revision : 0 Date : 990715 4. ANALYTICAL PROCEDURES 4.1 Sample pre-treatment Meat and liver (1 kg) is crushed and homogenised in a Moulinette S (Moulinex). A test portion of 5.0 g is weighted into a 50 ml centrifuge tube, spiked with a mixture of internal standards (a-zearalanol-d4 and b-zearalanol-d4) at the level of 10 ng (corresponding to 2 ng/g). 4.1.1 Meat samples For digestion, 10 ml volume of Trisbuffer (2.1.13) and 5 mg subtilisin (2.1.15) is added. The mixture is vortexed for 30 seconds and shaken thoroughly for two hours at 50o C. 4.1.2 Liver samples For hydolysis, 40 ml of Suc Helix Pomatia (2.1.11), diluted with 10 ml 2 M acetate buffer (pH 5.2) (2.1.12) is added. The mixture is vortexed for 30 sec., check pH 5.2 (when necessary adjust the pH with acetic acid to 5.2) and hydrolyse overnight at 37oC, with constant shaking by means of an incubator equipped with a shaker. 4.2 Liquid/liquid extraction To the mixture (4.1) 2 ml conc. HCl and 10 ml TBME is added, shake for 10 minutes and centrifuge for 3 minutes at 3600 rpm. The organic layer is collected in a glass tube. This operation is repeated with 5 ml TBME. The extract in the glass tube is evaporated at 50oC under a gentle stream of nitrogen.
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