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

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 [H-3] 19-nortestosterone laurate (19-NTL; 4-estren-17 beta-laurate-3-one) o r [H-3] 19-nortestosterone (19-NT; 4-estren-17 beta-ol-3-one; nandrolone). Hepatocyte medium was extracted with solid phase Clx media and analysed by narrow bore radio-HPLC-MSn (LCQ, Finnigan) to evaluate the structure of met abolites of 19-NTL and 19-NT. Radio-HPLC of hepatocyte medium extracts foll owing incubation with [3H]19-NTL confirmed that the first step of biotransf ormation 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 po larity. Hydrolysis of hepatocyte medium extracts with P-glucuronidase (Heli x pomatia) indicated that some of these metabolites were glucuronide or sul fate conjugates. Structural analysis of unconjugated metabolities by positi ve-ion atmospheric pressure chemical ionisation MS2 and comparison with ava ilable reference preparations indicated biotransformation of 19-NT to 4-est ren-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, 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 electr ospray ionization, which revealed the presence of glucuronide conjugates of alpha-(trace) and P-epimers of 19-NT, n-hydroxy-4-estren-3, 17-dione, n-hy droxy-4-estren-17-ol-3-one and 5 beta-estran-3 alpha,17 beta-diol. These st udies provide a clear indication of the route of hepatic metabolism in the bovine, which may now be readily substantiated by reference to samples, suc h as urine or bile, derived from animals treated with unlabelled 19-NTL.