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

Abstract

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-17β-laurate-3-one) or [³H]19-nortestosterone (19-NT; 4-estren-17β-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 β-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-17α-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, 5β-estran-3α-ol-17-one (noretiocholanolone) and 5β-estran-3α,17β-ol (major metabolite after 4 h). Conjugated metabolites were analysed by electrospray ionization, which revealed the presence of glucuronide conjugates of α- (trace) and β-epimers of 19-NT, n-hydroxy-4-estren-3,17-dione, n-hydroxy-4-estren-17-ol-3-one and 5β-estran-3α,17β-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.