Assay for trenbolone and its metabolite 17 alpha-trenbolone in bovine urine based on immunoaffinity chromatographic clean-up and off-line high-performance liquid chromatography-thin-layer chromatography.

An high-performance liquid chromatography (HPLC)-thin-layer chromatography (TLC) method was developed to detect the illegal use of the xenobiotic growth promotor Trenbolone acetate (TBA). Very effective clean-up of bovine urine was achieved by immunoaffinity chromatography (IAC). The active form of TBA, the steroid 17 beta-Trenbolone (17 beta-TB), as well as its major metabolite 17 alpha-Trenbolone (17 alpha-TB), were assayed simultaneously with HPLC and on-line UV detection. The fraction containing 17 alpha-TB and 17 beta-TB (TB-fraction) was collected, and for confirmation 17 beta- and 17 alpha-TB were subsequently separated and identified by TLC. The limit of detection by on-line HPLC-UV (350 nm) was 1-2 micrograms TB/l. Off-line TLC detection was even more sensitive, 0.5 microgram 17 beta- or 17 alpha-TB/1. The assay was validated by investigating urine samples from veal calves implanted with TBA. The presence of 17 beta- and 17 alpha-TB was clearly demonstrated. A survey of the illegal use of TBA in cattle was performed by applying the assay to urine obtained at slaughter. No residues of TBA or its metabolites were found in any of the 144 random samples from the Dutch public health surveillance programme.