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    Utility of isolated hepatocytes and radio-HPLC-MSn for the analysis of the metabolic fate of 19-nortestosterone laurate in cattle.

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    The metabolic fate of 19-nortestosterone laurate in cattle was investigated to evaluate target analyte(s) appropriate to surveillance for illicit use as a growth promoting agent. Bovine hepatocytes were incubated with either [3H]19-nortestosterone laurate (19-NTL; 4-estren-17 beta-laurate-3-one) or [3H]19-nortestosterone (19-NT; 4-estren-17 beta-ol-3-one; nandrolone). Hepatocyte medium was extracted with solid phase C18 media and analysed by narrow bore radio-HPLC-MSn (LCQ, Finnigan) to evaluate the structure of metabolites of 19-NTL and 19-NT. Radio-HPLC of hepatocyte medium extracts following incubation with [3H]19-NTL confirmed that the first step of biotransformation in liver was hydrolysis of the fatty acid ester to release [3H]19-NT, which, in turn, was converted into a range of metabolites of diverse polarity. Hydrolysis of hepatocyte medium extracts with beta-glucuronidase (Helix pomatia) indicated that some of these metabolites were glucuronide or sulfate conjugates. Structural analysis of unconjugated metabolities by positive-ion atmospheric pressure chemical ionisation MS2 and comparison with available reference preparations indicated biotransformation of 19-NT to 4-estren-17 alpha-ol-3-one, 4-estren-3, 17-dione (major metabolite after 1 h), n-hydroxy-4-estren-3, 17-dione, n-hydroxy-4-estren-17-ol-3-one, 5 beta-estran-3 alpha-ol-17-one (noretiocholanolone) and 5 beta-estran-3 alpha, 17 beta-ol (major metabolite after 4 h). Conjugated metabolites were analysed by electrospray ionization, which revealed the presence of glucuronide conjugates of alpha-(trace) and beta-epimers of 19-NT, n-hydroxy-4-estren-3, 17-dione, n-hydroxy-4-estren-17-ol-3-one and 5 beta-estran-3 alpha, 17 beta-diol. These studies provide a clear indication of the route of hepatic metabolism in the bovine, which may now be readily substantiated by reference to samples, such as urine or bile, derived from animals treated with unlabelled 19-NTL

    Metabolism of tilmicosin by rabbit liver microsomes and hepatocytes

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    We investigated tilmicosin (TIM) metabolism, at 25, 50 or 100 microM, in cultures of primary hepatocytes from rabbits bred commercially for food and in liver microsomes prepared from both untreated and rifampicin (RIF)-treated rabbits. RIF is a well-known cytochrome P4503A (CYP 3A) inducer in rabbits and most macrolides are known to be substrates of CYP 3A. No peaks in addition to those of the cis and trans forms of TIM were observed by high performance liquid chromatography (HPLC) in extracts of microsomes from untreated rabbits. When TIM was incubated with induced microsomes, at least two peaks were found by HPLC and an additional peak, eluting at shorter retention time was isolated from hepatocytes incubated for 24h with the macrolide. The structures of the metabolites were then estimated by liquid chromatography-mass spectrometry (LC-MS) in concentrated extracts from induced microsomes. Five metabolites were separated and putatively identified: cis and trans demethylated tilmicosin, tilmicosin N-oxide and cis and trans tilmicosin epoxide. The overall amount of metabolites produced in vitro using livers of untreated and RIF treated rabbits was very low, has also been observed in vivo and in vitro in cattle, chickens and pig

    In vitro metabolism of Clenbuterol and Bromobuterol using microsomes from swine liver

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    1. Clenbuterol (CBL) and bromobuterol (BBL) were both extensively metabolized by hepatic microsomes of swine to only one polar metabolite which was separated by hplc and purified to perform mass analysis. 2. LC-MIS analysis by direct infusion into an ion trap system and after reverse-phase chromatograpy into a triple quadrupole system showed that the metabolites were the hydroxylamine-derivatives of CBL and BBL. GC-MS analysis by the CI and EI modes confirmed that the hydroxyl group was bound to the aniline nitrogen. The chemical instability of those metabolites probably as a consequence of spontaneous oxidation and reduction gave rise during the analysis to the corresponding nitroso and nitro derivatives, together with the original compound. 3. Thermal inactivation and CO complex formation were used selectively to inactivate flavin monooxygenase and cytochrome P450, respectively. Both inactivation procedures significantly reduced the formation of the hydroxyl metabolite

    Doxycycline and sulfadimethoxine transfer from cross-contaminated feed to chicken tissues.

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    During feed preparation at feed mills or during feed mixing in bins at farms, the accidental contamination of feed at trace levels by veterinary drug residues, commonly known as carry-over, can accidentally but frequently occur. To evaluate the concentrations of residual antimicrobials in poultry edible tissues, due to contaminated feed, sulfadimethoxine and doxycycline were administered for 10 days to chickens in poultry feed incurred at the contamination levels frequently found during national feed monitoring programmes (1-5 mg kg(-1)). Sulfadimethoxine and doxycycline residual concentrations detected in muscle (<LOD and 31 μg kg(-1), respectively), liver (13 and 56 μg kg(-1), respectively) and kidney (56 and 115 μg kg(-1), respectively) were compared with their maximum residue limits (MRLs) fixed by EC 470/2009 and EU 37/2010 Regulations for a preliminary risk evaluation

    Going Beyond Counting First Authors in Author Co-citation Analysis

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    The present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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