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 17 alpha-trenbolone (17 alpha-TbOH), 4-chloroandrost-4-ene-3,17-dion (CLAD), 16- beta -OH-stanozolol (16OHstan) and alpha- and beta-boldenone (alpha -Bol, beta -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 C(18) 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 CC beta smaller then 1 ng/ml was obtained for each compound.