The illegal use of nandrolone) and its esters in livestock, for growth promotion purposes, has been widely reported in the European Union. The target residues for surveillance of abuse in bovine urine and bile samples are 17a-and 17b-19NT, although this choice of target residues is not based on in vivo radiotracer biotransformation data. In this study, four steers were administered [ 3 H 2 ]-and [ 2 H 3 ] 17b-19NT laurate (2 mg kg 21 body mass) by intramuscular injection and blood, urine, faeces and bile samples were taken for 30 d until slaughter, after which tissues were sampled for total residue analysis. Total plasma radiolabelled residues reached a maximum of 56.3 ± 15.9 pmol ml 21 at 36 h and were still appreciable (13.3 ± 1.6 pmol ml 21 ) 30 d after treatment. Throughout the study period, total residue concentrations in bile (about 2-16 nmol ml 21 ), urine and faeces (0.5-3 nmol ml 21 or g 21 ) were higher than in other tissues sampled at slaughter. At slaughter there was evidence of residue accumulation in pigmented eye tissue (33.1 ± 6.1 pmol g 21 ) and in white (13.4 ± 3.4 pmol g 21 ) and black hair (28.9 ± 8.9 pmol g 21 ). Evaluation of radio-HPLC profiles of urine and bile extracts generally indicated that 19NT and 19NT laurate residues were present in relatively small amounts among a complex mixture of metabolites. GC-MS analysis of glucuronidase-hydrolysed bile extracts indicated that the major metabolites were 5b-estrane-3a,17a-diol, 5a-estrane-3b,17a-diol, 5a-estran-3a-ol-17-one (norandrosterone) and estra-1,3,5(10)-triene-3,17a-diol (17a-estradiol).

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.