Metabolism of a sea lamprey pesticide by fish liver enzymes part B: method development and application in quantification of TFM metabolites formed in vivo.
The sea lamprey (Petromyzon marinus) is a destructive invasive species in the Great Lakes. Since the 1960s, tons of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) has been applied to selected tributaries each year to eliminate or reduce sea lamprey larval populations. Therefore, the environmental impact of TFM needs to be evaluated. However, the metabolism of TFM and its mechanism of selective toxicity in sea lamprey is not yet fully understood. Based upon our previous report on the identification, synthesis, and characterization of TFM metabolites observed in liver incubates from sea lamprey and non-target fishes, we now provide a robust assay for quantifying TFM and its metabolites in fish liver tissue. This method is important for assessing bioaccumulation of TFM in the ecosystems. The compounds purified in our previous report were used to develop and validate a quantitative ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for TFM and TFM metabolites formed in vivo. Several sample preparation techniques were compared, and a protein precipitation method was selected. The unavailability of stable isotopic internal standards was overcome by using a matrix matching method. After a thorough validation, this method was applied to determine the concentrations of TFM and its metabolites in fish liver tissues from animals exposed to TFM, and in the comparison between dead animals and survivors. Seven of eight expected metabolites were observed, some for the first time in vivo. Our results indicate that in vivo nitroreduction, glucuronidation, sulfation, and glutathione conjugation are involved in TFM metabolism in sea lamprey.