Analysis of trenbolone acetate metabolites and melengestrol in environmental matrices using gas chromatography-tandem mass spectrometry.

Studies demonstrate that exposure to steroid hormones in receiving waters can adversely impact reproduction of aquatic organisms. In particular, exogenous steroid hormones widely used as growth promoters in animal agriculture are of high concern, yet no gas chromatography-tandem mass spectrometry (GC/MS/MS) analytical methods for the detection of these compounds in complex environmental matrices is described in the literature. This study utilizes analytical methods based upon N-methyl-N-(trimethylsilyl)trifluoro-acetamide-iodine (MSTFA-I(2)) derivatization for the analysis of metabolites of trenbolone acetate (TBA), including 17α-trenbolone, 17β-trenbolone, and trendione, and melengestrol acetate in receiving waters and surface soils associated with animal agriculture. Results suggest method detection levels of 0.5-1 ng/L for the trenbolone metabolites, while detection of melengestrol is qualitative only. Isotope dilution methods employing d3-17β-trenbolone were used to improve steroid quantification. Method recoveries in spiked samples collected from a variety of representative receiving waters generally ranged from 80-120% with consistent and low standard deviation (generally<10%) for replicate analysis. Analysis of a storm water runoff sample from a commercial confined animal feeding operation (CAFO) that used TBA implants detected 17β-trenbolone and trendione at concentrations of 31 and 52 ng/L, respectively. Analysis of surface soils at a commercial CAFO using TBA implants detected 17α-trenbolone at concentrations between 4-6 ng/g dry weight. Method development efforts suggested that the concentration of I(2) in MSTFA, the removal of I(2) from sample extracts after derivatization, and the use of Florisil clean-up to reduce organic matter matrix were vital aspects of steroid hormone quantification at low (<30ng/L) concentrations in complex environmental matrices.