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Developing an Untargeted High-resolution Mass Spectrometry Method for the Detection and Identification of Glucuronide Biotransformation Products in Environmentally Exposed Fish
Citation:
Evich, M., J. Mosley, I. Ntai, D. Ekman, AND T. Collette. Developing an Untargeted High-resolution Mass Spectrometry Method for the Detection and Identification of Glucuronide Biotransformation Products in Environmentally Exposed Fish. 2019 American Society for Mass Spectrometry (ASMS), Atlanta, GA, June 02 - 06, 2019.
Impact/Purpose:
Environmental contaminants can be prioritized by detecting and identifying glucuronidated biotransformation products in fish bile using untargeted mass spectrometry-based metabolomics.
Description:
Introduction The complexity of contaminant mixtures in surface waters has made traditional assessments of risk to resident species (e.g., fish) through targeted methods increasingly difficult. In addition to the hundreds of targeted chemicals detected in surface waters, nontargeted methods are uncovering many more contaminants that could potentially cause adverse biological impacts. An initial screen to prioritize contaminants should include bioavailability, assuming only those bioavailable contaminants may pose potential risks. Glucuronidation, the enzymatic attachment of a glucose moiety to a xenobiotic, is a major metabolic pathway of detoxification, and thus an important indicator of bioavailability. We have developed a novel untargeted method to detect glucuronides in bile of environmentally exposed fish to identify bioavailable contaminants from environmental mixtures. Methods Identifying glucuronides in an untargeted manner relies on a traditional biological assay coupled with untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS). Briefly, gallbladder extracts from fish exposed to wastewater treatment plant (WWTP) effluent were split into two aliquots, one of which was hydrolyzed with β-glucuronidase. The other portion was treated as a paired control. After digestion, the reaction was quenched using methanol, filtered, reconstituted and underwent untargeted LC-MS acquisition (Orbitrap Q-Exactive) followed by data analysis with Compound Discoverer for the putative identification of glucuronidated xenobiotics. Untreated bile samples were analyzed in parallel by structure-specific neutral loss-dependent MS3 fragmentation using an Orbitrap ID-X Tribrid mass spectrometer to confirm the presence of a glucuronide and aid identification of the parent xenobiotic. Preliminary Data Previous untargeted analyses of the skin mucus and liver metabolomes from fathead minnows exposed to WWTP effluent suggested the presence of biotransformation products. The detection of glucuronidated BPA in the skin mucus of these same fish provided additional evidence that xenobiotic transformation was induced upon exposure to the effluent. These results led to the development of this novel untargeted method for detecting xenobiotic glucuronides in blood plasma and bile, the latter of which is generally considered to have the highest concentrations of glucuronides. The key to identifying xenobiotics taken up and glucuronidated by the fish is to look for analytes that are up- or down-regulated in the hydrolyzed aliquot relative to the paired control due to the removal of the glucuronide moiety in the hydrolysis step. Preliminary results revealed that the highest up-regulated analytes from untargeted LC-MS analysis were deconjugated xenobiotics (both from spiked standards and untargeted compounds), whereas the most significant down-regulated analytes were xenobiotic glucuronide standards spiked into human plasma extracts. Furthermore, as a confirmatory method, structure-specific neutral loss-dependent MS3 fragmentation using an Orbitrap ID-X Tribrid mass spectrometer was applied to fish bile extracts spiked with xenobiotic glucuronide standards. In brief, characteristic neutral loss and product ions during fragmentation are used as handles to further elucidate the glucuronidated contaminants. Preliminary data analysis was performed with a new workflow developed within Mass Frontier. For each xenobiotic glucuronide standard, the correct de-conjugated parent compound was returned as the top hit from a substructure search within the mzCloud online database. Novel Aspect Environmental contaminants can be prioritized by detecting and identifying glucuronidated biotransformation products in fish bile using untargeted mass spectrometry-based metabolomics.