Persistance of illegal drugs in bovine manure

Oral or parenteral livestock drugs can be detected unmodified or as metabolites in animal dejections (urines and/or faeces). During manure maturation and before its disposal on cultivated lands, drugs present in cattle dejections can undergo a degradation, most of all due to the presence of faecal bacteria. The degradation level depends on the chemical structure of the different compounds. The general purpose of this work is to evaluate if manure can be a useful matrix to detect the presence of forbidden drugs in animal production, so to become a cheap and quick method of analysis. Considering the compounds banned in European Directive 96/23/EC group A and in attachment IV of Reg. CEE 2377/90, some of them have been chosen, based on positive cases frequency in later years, to select those of most interest for cattle husbandry. A bibliographic research has been performed on these compounds, to analyze their metabolic characteristics and to find out the best analytical methods to check these substances during screening and confirmatory analysis. Methods have been improved for screening analysis, using immunoassay techniques with sensitivity more or less up to ng/g. Generally we have adapted kits ELISA, already used in urine or in other tissues, to detect these compounds in manure. We optimized in manure matrix HPLC MS-MS methods for compounds where kit ELISA could not be used and to confirmatory analysis. Afterwards, the degradation kinetics of substances in manure has been evaluated in a 4 months period, using in vitro models and both ELISA and HPLC MS-MS techniques. The studied substances and preliminary results are reported below. STANOZOLOL • ELISA technique: a kit for urine, serum and plasma has been adapted. We have found the limit of quantification about at 0.1ng/ml and the curve saturation was at 2ng/ml. Matrix effect was high. • HPLC MS-MS: the method has been optimized for 16 β OH stanozolol with CCα of 0.42ng/ml, CCβ of 0.58ng/ml. ZERANOL α and β • ELISA technique: a kit for urine, serum and bovine tissue has been adapted. Matrix effect was high so the maximum level for quantification was 0.67ng/ml. • HPLC MS-MS: the method has been optimized to show both of the compounds, CCα was 0.25ng/ml and 0.45 ng/ml for α and β-zeranol respectively; CCβ was 0.45ng/ml and 0.90ng/ml for α and β zeranol respectively. NITROFURAN (AOZ and AMOZ) • ELISA technique: a kit for different tissues in different animal species has been adapted. Matrix effect was scarce for AOZ but not so waek for AMOZ. Both metabolites showed sensibility close to 0.2ng/ml. • HPLC MS-MS: samples have been derivatized before extraction. CCα was 0.20ng/ml and CCβ 0.30ng/ml for AMOZ and 0.45ng/ml and 1.30ng/ml for AOZ. Β2 AGONISTS (CLENBUTEROL and TERBUTALINE) • ELISA technique: a kit for analyzing clenbuterol and another one for analyzing together clenbuterol and terbutaline have been adapted. The limit of quantification was between 0.1 and 0.2ng/ml and saturation level about at 10ng/ml for clenbuterol on its own; together with terbutaline the limit of quantification was 0.5ng/ml and saturation level between 10 and 20ng/ml without matrix effect. • HPLC MS-MS: CCα was 0.14ng/ml and CCβ was 0.22ng/ml. DIETHYLSTILBESTROL • ELISA technique: a kit for urine, bile, muscle and faeces has been adapted. The limit of quantification was between 12.5 and 25ppt, the saturation level about on 10ng/ml without matrix effect. • HPLC MS-MS: CCα was 0.41ng/ml and CCβ was 0.86ng/ml. TRENBOLONE • ELISA technique: a kit for urine, bile, muscle, liver and faeces has been adapted. The limit of quantification was 2ng/ml and saturation level of 10ng/ml • HPLC MS-MS: CCα was 0.80ng/ml and CCβ was 1.10ng/ml. CHLORANPHENICOL • ELISA technique: a kit for honey, eggs, urine, milk, plasma, meat and fish has been adapted. The limit of quantification was between 0.2 and 0.5ng/ml • HPLC MS-MS: CCα was 0.32ng/ml and CCβ was 1.07ng/ml. ISOXSUPRINE • HPLC MS-MS: we searched this compound only with mass spectrometry and CCα and CCβ values were 0.24ng/ml and 0.36ng/ml respectively. TIOURACIL • HPLC MS-MS: it has been derivatized before extraction; we found 0.34ng/ml for CCα and 0.49ng/ml for CCβ. CORTICOSTEROIDS • HPLC MS-MS :we searched some drugs: prednisolone, prednisone, cortisol, cortisone, dexamethasone, betamethasone and methylprednisone and we found CCα from 0.10ng/ml to 0.60ng/ml and CCβ from 0.15ng/ml to 0.75ng/ml for all compounds. ANABOLIC-ANDROGENIC STEROIDS • HPLC MS-MS: we searched androstadienedione, α-boldenone, β-boldenone, androstenedione, testosterone and epitestosterone only with mass spectrometry. If we exclude ET CCα value range from 0.20ng/ml to 0.80ng/ml and CCβ from 0.40ng/ml to 2.50ng/ml