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Analytical Method Development for the Detection of the Metabolites of the Hormonal Steroids Trenbolone Acetate and Melengestrol Acetate in Complex Environmental Matrices using Gas Chromatography / Tandem Mass Spectrometry
AuthorParker, Jed A.
AdvisorKolodziej, Edward P.
Civil and Environmental Engineering
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The occurrence of hormonal steroids originating from cattle implanted with anabolic growth promoters in surface waters is of great consequence to sensitive aquatic species. Exposure to excreted metabolites of trenbolone acetate, the most prevalently used growth promoter in the U.S., has been shown to have deleterious, androgenic effects on sensitive aquatic species, such as decreased fecundity and altered secondary sexual characteristics. The potential risk of this exposure is population collapse in isolated, sensitive populations. These effects have been observed when exposure concentrations to trenbolone metabolites are as low as 0.3 ng/L. In light of observed effects on aquatic organisms, a number of analytical techniques have been developed to detect trenbolone metabolites in environmental matrices. Such techniques include bio-assays and liquid or gas chromatography paired with mass and tandem mass spectrometry. While there are existing liquid chromatography / tandem mass spectrometry methods, to date, there are no gas chromatography / tandem mass spectrometry methods which detect the trenbolone acetate metabolites, 17α-trenbolone, 17β-trenbolone and trendione in environmental matrices. The research presented describes such an analytical method for environmental matrices.The analytical method, presented here, draws on gas chromatography paired to mass spectrometry and tandem mass spectrometry methods developed for 17α- and 17β-trenbolone detection in biological matrices, and extends them to more complex environmental matrices. In addition, this method attempts to incorporate the third trenbolone acetate metabolite, trendione and the melengestrol acetate metabolite, melengestrol. Trendione was added to complete the family of trenbolone acetate metabolites, allowing for the possibility of conducting fate and transport studies. Melengestrol was added to the method to examine the possibility of utilizing the established MSTFA/I<sub>2</sub> derivatization procedure for other classes of steroids such as progestagens. Results indicate that 17α- and 17β-trenbolone can be recovered from environmental matrices with excellent chromatographic response at or below a concentration of 2.5 ng/L. Trendione, owing to the possible degradation of the derivatized product, has shown more difficulty with recoveries in environmental matrices. It is theorized that stabilizing trendione after derivatization or analyzing samples within 48 hours will allow for recoveries similar to that of 17α- and 17β-trenbolone. Attempts at matrix spike recoveries with melengestrol limited its detection in environmental matrices to 100 ng/L. An observed standard concentration of 20 µg/L was successfully analyzed for melengestrol which leads to the possibility that it can be analyzed at much lower concentrations within the GC/MS/MS. As with trendione, if a stable derivatization product can be formed for melengestrol, it is expected that it can be recovered in environmental matrices at relevant concentrations of 2.5 ng/L and below.