Screening of clostebol and its metabolites in bovine urine with ELISA and comparison with GC-MS results in an interlaboratory study

An extraction procedure for clostebol metabolites in urine is developed including enzymatic hydrolysis of conjugated metabolites with Helix pomatia juice (SHP) and solid-phase extraction (SPE) with further cleanup of sample extracts. For the enzymatic deconjugation step, variables such as buffer pH, amount of enzyme, incubation time, and temperature are examined. For the SPE step, different wash solutions and combinations with subsequent liquid-liquid extractions are examined. Incurred bovine urine samples, obtained through oral and intramuscular administration of clostebol acetate to animals, are used to test the performance of the developed method. In addition to the optimization of the sample pretreatment procedure, an interlaboratory study for the analysis of the incurred urine samples with ELISA and GC-MS is performed and good agreements are observed.