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Abstract
A simple and sensitive detection method based on C18 reversed-phase HPLC with on-line UV-detection at 350 nm is presented for screening the xenobiotic steroid, trenbolone, and its major metabolite, epi-trenbolone, in bovine liver and muscle. On-line high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS) methodology is used for confirmation of trenbolone residue in liver extracts. Trenbolone was extracted from bovine liver and muscle using three-phase liquid-liquid extraction which included a mixture of water, acetonitrile, dichloromethane, and hexane. The target compounds are extracted from tissue into the acetonitrile layer. The residue from this extraction was then subjected to solid phase extraction using C18 and silica disposable cartridges with methanol-water and benzene-acetone as eluents. Screening for trenbolone is then done by reversed-phase HPLC followed by LC/MS/MS confirmation under selected reaction monitoring (SRM) conditions. A structural analog of trenbolone, 19-nortestosterone, was chosen as the internal standard for quantification by HPLC. HPLC screening can be accomplished at the 0.5 ppb level in bovine liver and muscle extracts. Confirmation can be accomplished at the 1.0 ppb level by LC/MS/MS.