Identification and quantitation of trenbolone in bovine tissue by gas chromatography-mass spectrometry

Identification and quantitation of trace amounts of trenbolone in bovine tissue by capillary gas chromatography-mass spectrometry-selected-ion monitoring (GC-MS-SIM) has been developed. Three-phase liquid-liquid extraction using a mixture of water-acetonitrile-dichloromethane-hexane was utilized for the sample extraction from tissue. Target compounds were extracted from the tissue into the acetonitrile layer. The residue from this extraction was then subjected to solid-phase extraction by C18 and silica gel disposable cartridges using methanol-water and benzene-acetone as eluents. To overcome extensive matrix interferences, preparative reversed-phase high-performance liquid chromatographic separation was used with an octadecyl-bonded column using methanol-water as mobile phase for sample clean-up prior to GC-MS analysis. A structural analogue of trenbolone, 19-nortestosterone, was chosen as the internal standard for quantitation by GC-MS. The sample was co-injected with N,O-bis (trimethylsilyl) trifluoroacetamide-1-(trimethylsilyl) imidazole (95:5, v/v) for flash heater derivation. Identification and quantitation were simultaneously carried out by SIM of characteristic ions of the trimethylsilyl derivatives of trenbolone and 19-nortestosterone. The limit of detection for trenbolone and epitrenbolone was 0.5 ppb in muscle and liver tissue. A comparison of sensitivity and specificity between GC-MS under electron ionization in addition to positive- and negative-ion chemical ionization conditions using methane reagent gas is also discussed.