Development and validation of a multi-analyte method for the detection of anabolic steroids in bovine urine with liquid chromatography–tandem mass spectrometry

Detection of anabolic steroids in animal urine samples is currently performed with GC–MS in our lab. However we found that the detection of 17α-trenbolone (17α-TbOH), 4-chloroandrost-4-ene-3,17-dion (CLAD), 16-β-OH-stanozolol (16OHstan) and α- and β-boldenone (α-Bol, β-Bol) was very difficult, if not impossible. Therefore a sensitive, specific and selective qualitative multi-analyte LC–MS–MS method was developed. The LC separation was achieved by using a Symmetry® C18 column and methanol–water–formic acid (54.7–44.7–0.6) as a mobile phase at a flow-rate of 0.3 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode with positive electrospray interface. Validation of the method was done according to draft SANCO/1805/2000 Rev.1 and a CCβ smaller then 1 ng/ml was obtained for each compound.