Screening and confirmatory GC-MS methods for the detection of trenbolone in bovine urine

Sensitive and selective methods for the screening (GC-MS) and confirmatory analysis (GC-MS/MS) of 17α- and 17β- trenbolone in bovine urine were developed. In the first stage of the analysis, the enzymatic hydrolysis of trenbolone metabolites with glucuronidase AS-HP in acetate buffer (pH 5.2) solution was carried out. Free compounds were extracted from urine with diethyl ether. For the purification of the extract solid phase, extraction with C18 and NH2 columns was applied. The evaporated extract was subjected to two derivatisation steps; the first with MSTFA/I2 solution and second with MSTFA. The separation of the analytes on HP-5 ms capillary column was conducted. The methods were validated according to the Commission Decision 2002/657/EC. For GC-MS method, CCα and CCβ were 0.21-0.36 μg L⁻¹ for 17α- trenbolone and 0.20-0.34 μg L⁻¹ for 17β- trenbolone, while for GC-MS/MS method the values were lower and amounted to 0.15-0.25 μg L⁻¹ for 17α- trenbolone and 0.20-0.34 μg L⁻¹ for 17β-trenbolone. Method recoveries in spiked samples ranged from 86%-111% with standard deviation lower than 25% for both detection techniques.