Development and Validation of a Screening Method for DES, Zeranol ...

Journal of Analytical Toxicology, Vol. 22, September 1998

Development and Validation of a ScreeningMethod for DES, Zeranol, and 13-Zearalanolin Bovine Urine by HRGC-MS and Evaluationof Robustnessfor Routine Surveyof the Brazilian Herd Marlice A.S. Marques*, tucilene A. Lima, Carlos H.B. Bizarri, Francisco R. Aquino Neto, and Jari N. Cardoso Departamentode Qufmica Org~nica, CT-BI. A-51. 607, Institutode Qufmica, UniversidadeFederaldo Rio de ]aneiro, Rio de Janeiro, 21949-900, Brasil

[Abstract [ A method and evaluated for screening and confirmation of diethylstilbestrol (DES), cr and [~-zearalanol in bovine urine was developed. The residues were extracted from urine by Cls cartridges and purified on alumina columns. For screening and confirmation purposes, the anabolic derivatives were analyzed by gas chromatography-mass spectrometry after derivatization with BSTFA + 1% TMCSor a solution of PFPMacetone (1:2, v/v), respectively. The recovery of most analytes for the whole procedure was higher than 96%, with a detection limit of 0.5 ppb. This procedure is being routinely applied to the Brazilian National Program for the Control of Residues in Meat (PNCRBC).

ties (1) prohibit the use of any kind of anabolic compounds in meat production. In order to control the illegal use of these drugs in Brazilian cattle, a new work-up procedure using solid-phase extraction (SPE) on silica C18 columns followed by a cleanup on alumina was evaluated and validated in our laboratory (6) for analysis of DES and ~- and [3-zearalanol in urine. The anabolic residues were determined by capillary gas chromatography-mass spectrometry (GC-MS) using stable-isotope DES (DES-d6) as internal standard.

Experimental Introduction

Equipment

Growth-promoting agents have been used in meat production for over 30 years. The production of meat protein is an inefficient process compared, for example, with the production of milk, eggs and poultry. The use of anabolic compounds can significantly improve this efficiency.Anabolic agents are defined as substances that increase nitrogen retention and protein deposition in animals (1,2). The effects of anabolic compounds are expressed in terms of the improvement of the average daily weight gain (DWG);feed conversion efficiency(FE); and carcass quality. The possible health effects of anabolic agents have been the subject of constant concern and discussion (3,4). The carcinogenicity in humans of one of the earliest anabolic compounds, diethylstilbestrol (DES), was set as an example of the dangers associated with use of exogenous compounds with hormonal activity (3,4). DEs was the first anabolic agent to be banned for use in meat production in Brazil and in other countries (1,2,5). Current Brazilian legislation (2) and the European Communi* Author to whom correspondenceshould be addressed.

Analyses were carried out on a Hewlett Packard (Palo Alto, CA) 5890 series II GC equipped with a 7673Aautomatic injector with electronic pressure control and interfaced to an HP 5972 mass selective detector. MS operating temperatures were as follows: transfer line, 300~ ion source, 250~ interface, 300~ and quadrupole, 120~ Detection was done by selected ion monitoring (SIM)with a dwell time of 40 ms. The ionisation was by electron impact at 75eV. GC operating conditions were as follows: injector, 250~ detector, 300~ column, 120~ (initial temperature, held for 1 rain); followed by a gradient of 20~ to a final temperature of 300~ (held 7 rain); flow rate, 0.59 mL/min; initial pressure, 30 psi; final pressure, 14.5 psi; average linear velocity (p), 22 cm/s; 1-2-pL samples were injected in the splitless mode. An HP-5 fused-silica capillary column (25 m x 0.2-ram i.d., 0.33-pro film thickness) was used. Materials

All reagents and solvents were of analytical-reagent grade. Water was doubly distilled. Solvents, acetone, ethanol, and methanol, were obtained from Merck (Darmstadt, Germany). Enzymes were 13*glucuronidase-sulfataseand Helixpomatia,

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Journal of Analytical Toxicology, Vol. 22, September 1998

containing 100,000 units [3-glucuronidase and 1,000,000 units sulfatase per milliliter (cat. no. 6707, Sigma, St Louis, MO). The acetate buffer was 2M and had a pH of 5.2. Derivatization reagents were N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)with 1% trimethylchlorosilane (TMCS; cat. no. T6381, Sigma) and pentafluoropropionic acid anhydride (PFPAcat. no. 65192M, Pierce, Rockford, IL) Reference compounds. DES, DES-monoglucuronide, and r162and [3-zearalanol used for this research were obtained from Sigma. Identity was confirmed by GC-MS. Standard solutions. All solutions were prepared in methanol. Stock solutions were prepared at concentrations of 1 mg/mL and stored in the dark at -20~ for up to a year. Working solutions were obtained by diluting stock solutions 1:100 and were stored in the dark at 4~ for up to three months. A standard mix solution containing DES and resorcyclic acid lacotones (RALs) at a concentration of 0.1 ng/pL in ethanol was prepared from the working solution and used for the fortification of samples. lnternalstandard. For purposes of quality control and accurate quantitation, a deuterated internal standard (IS) (DES-d6 donated by RIVM/ARO,Bilthoven, The Netherlands) was used. The purity of IS was checked by GC-MS, Fourier-transform infrared detection, and high-performance liquid chromatography (HPLC), and it was 98% deuterated. SPE columns. Cls columns (1 mL) were obtained from J.T. Baker (Phillipsburg, NJ). Aluminum oxide (neutral activity grade I) was purchased from Merck. Filtration columns (3 mL) obtained from J.T. Baker were filled with • 0.5 g of neutral alumina previously plugged with a frit; a second frit was put on top of the alumina layer. Sample. All urine samples used in this study were blanks from bovines created at a research veterinary farm (LANARKMinas Gerais, Brazil) without administration of anabolic compounds.

Cleanup with Cs8 and alumina columns. The same C18 column used for extraction of anabolics from matrix was reconditioned using I mL of methanol and 2 mL of water. After cooling the incubate to room temperature, the enzymatic digest was applied to the previously reconditioned C18 column. The washing step was done using 1 mL of water and 1 mL of methanol/water (50%, v/v) at a flow rate of 1 mL/min. Air was passed through the column to completely eliminate residual wash solvent. The analytes of interest were eluted with 1 mL of methanol at a flow rate of 1.0 mL/min. The solvent was completely evaporated under a stream of nitrogen at 50~ and the sample was dissolved in 300 pL acetone/water (95:5, v/v). The solution was vortex mixed and passed through an alumina column previously conditioned with 2 mL of acetone/water (95:5, v/v). The residues were eIuted from the column with 3 mL acetone/water (95:5, v/v). The eluate solvent was carefully evaporated under a stream of nitrogen at 50~ Derivatization. For screening and confirmation, 50 pL BSTFAwith 1% TMCS or 50 pL of a solution of acetone/PFPA (2:1, v/v) was added to the dry residue from the alumina column. The reaction mixture was vortex mixed for 30 s and maintained at 60~ for 1 h. The solvent was subsequently evaporated to dryness under a stream of nitrogen at 50~ The derivatives were taken up in 50 pL of isooctane, vortex mixed, and transferred immediately to an injection vial. One or two microliters was injected into the GC-MS system. Statistical analysis. The data obtained in these studies were statistically evaluated by analysis of variance (ANOVA)and weighted least-squares regression using a standard spreadsheet program (RIVM/ARO,Bilthoven, The Netherlands).

Results and Discussion

Procedure for the SPE method Extraction of anabolics from matrix. Five milliliters of urine A number of studies were undertaken to evaluate and validate was taken up and transferred to a 10-mL glass tube containing the most critical steps of the whole analytical procedure. All a previously established amount of IS (DES-d6)and the analytes GC-MS analyses were performed under identical conditions, as of interest. The tube was shaken for 30 s on a vortex-type mixer, detailed previously (see Experimental). and the urine was dispensed onto the C18column that was previously conditioned once with 1 mL of methanol and once with Derivatization 2 mL of water at a flow rate of 1.0 mL/min. The column was then Trimethylsilyl (TiVlS) derivatives. TMS derivatives were washed with 2 mL of water at a flow rate of 1.0 mL/min to remove urine pigments. Table I. Mass-to-Charge Ratio of Diagnostic Ions Resulting from Electron Impact The residues of interest were eluted from Ionization (El) of the Anabolic Derivatives Investigated the C18 column with 1 mL of methanol, and the solvent was reduced to approxiCompound AbbreviationsMo' Derivative Md* Ions(m/z) mately 100 pL under a stream of nitrogen Diethylstilbestrol DES 268 TMS 412 412, 397, 383 at 50~ The residue was taken up in 4 PFP 560 560,531,397, 367, 291 mL of water and transferred to a 10-mL Diethylstilbestrol-d6 DES-d 6 268 TMS 413 418,403,389 tube for enzymatic deconjugation. PFP 566 566,533,403, 370, 294 Enzymatic &conjugation. One ~.-Zeranol ~-ZER 322 TM5 538 538,523, 433, 335, 307 milliliter of 2M acetate buffer (pH 5.2) PFP ~14 614,596, 525, 511,468, 455, 4~4, 308 and 50 gL of enzyme were added to the ~Zearalanol ~-ZER 322 TMS 538 538,523,433, 335, 307 dissolved extract (4 mL) previously obPFP 614 614,596, 525,511,468, 455,414, 308 tained. The solution was carefully shaken * Mo = Molecular mass. for about 30 s, and the homogenate was ~" M d -- Molecular mass after derivation. incubated for 2 h at 38~ 368

Journal of Analytical Toxicology, Vol. 22, September 1998

selected for the screening analysis of anabolics (Table I and Figure 1). Pentafluoropropronic (PFP) derivatives. For confirmation of anabolics in real samples, PFP derivatives were preferred because of better chromatographic characteristics relative to

TMS derivatives (e.g., elution time after most chromatographic interferences; a combination of high masses, large abundances, and many significant fragments, allowing higher sensitivity and more specific detection on GC-MS analyses by electron impact). (Table I and Figure 1). Average of 12.390 to 12.436min.: 13M665.D(*)

Average of 7.522 to 7.595 min.: 15M270.D (*)

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