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

Abstract

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 17β-Trenbolone (17β-TB), as well as its major metabolite 17α-Trenbolone (17α-TB), were assayed simultaneously with HPLC and on-line UV detection. The fraction containing 17α-TB and 17β-TB (TB-fraction) was collected, and for confirmation 17β- and 17α-TB were subsequently separated and identified by TLC. The limit of detection by on-line HPLC-UV (350 nm) was 1–2 μg TB/1. Off-line TLC detection was even more sensitive, 0.5 μg 17β- or 17α-TB/1. The assay was validated by investigating urine samples from veal calves implanted with TBA. The presence of 17β- and 17α-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.