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Untargeted MS-based Monitoring of Glucuronides in Fish: Screening Complex Contaminant Mixtures for Biological Relevance
Citation:
Mosley, J., M. Evich, I. Ntai, D. Ekman, Dan Villeneuve, G. Ankley, AND Tim Collette. Untargeted MS-based Monitoring of Glucuronides in Fish: Screening Complex Contaminant Mixtures for Biological Relevance. 2020 Sanibel Conference, Captiva Island, FL, January 23 - 26, 2020.
Impact/Purpose:
Poster presented at the 2020 Sanibel Conference.
Description:
INTRODUCTION. The complexity of contaminant mixtures in surface waters has made traditional assessments of risk to public health and the environment increasingly difficult, especially where toxicity information is unknown. Hence, new methods are needed to prioritize potential toxicants, which can be present below detection limits or missing from targeted analytical lists. Bioavailability, which is monitored by tracking both parent or metabolites of chemicals in resident environmental species (e.g., fish), can be an initial screen for the potential for toxicity. In particular, glucuronidation, which is a major pathway of detoxification, consists of a glucuronic acid moiety attached to the substrate, which facilitates eventual excretion of the xenobiotic. Thus, we investigated glucuronidation in fish exposed to treated wastewater effluent as a way to prioritize potential toxicants. METHODS. A novel high-resolution accurate mass spectrometry (HRAM-MS)-based method for untargeted glucuronide identification in biofluids was developed. The method was applied to bile from fathead minnows (Pimephales promelas) exposed for 21 d to treated wastewater effluent. Gallbladders were homogenized and spiked with isotopically labelled glucuronide standards. Aliquots were set aside for HRAM-MSn analysis (n ≤ 3), focusing on neutral loss (HRAM-MS2; +ESI and −ESI), glucuronide characteristic fragment ion (HRAM-MS2; −ESI) and structure-specific neutral-loss dependent (HRAM-MS3; +ESI) spectral acquisition. Remaining gallbladder extracts were split into paired samples for hydrolysis and spiked with buffered β-glucuronidase (enzyme fractions; +E) or buffer only (paired controls; −E). Both +E and −E fractions were analyzed with HRAM-MS1 spectral acquisition for traditional confirmation of glucuronidation via enzyme hydrolysis. PRELIMINARY DATA. Method validation with authentic standards revealed the presence of seven characteristic glucuronide fragment ions in −ESI mode. Their abundance varied widely depending on the parent substrate (e.g., oxazepam vs dihydrocodeine), though the most consistent fragment was at m/z 113.0244. Additionally, a neutral loss of 176.0321, corresponding to the loss of glucuronide, was present for all standards. A minimum compound class score (developed from authentic standards) revealed 173 glucuronidated compounds present in (unhydrolyzed) gallbladder extracts. Interestingly, only 73 out of 173 glucuronides were confirmed with enzymatic hydrolysis, suggesting that the majority of glucuronide substrates remained unhydrolyzed (or only partially hydrolyzed) after incubation with β-glucuronidase for X hrs. Within the compound class, 102 glucuronides were designated as xenobiotic (71 as endogenous) based on their absence (or presence) in gallbladder extracts from fish exposed to control water. Metabolite identification resulted in the putative annotation of 90 glucuronidated substrates in gallbladder extracts of fish exposed to treated wastewater effluent. Of these, only the endogenous glucuronide of testosterone was annotated by matching the MS2 spectrum to that of testosterone glucuronide in mzCloud, highlighting the lack of MS2 spectra for glucuronides in the database. Furthermore, only three additional glucuronide substrates (two of which were xenobiotic) were identified using the Identity Substructure algorithm, which compares the experimental MS3 spectrum of the deconjugated substrate to the MS2 database entry for the parent substrate. The remaining 86 glucuronide substrates were annotated by MSn (n ≤ 3) structural elucidation from hits found in Chemspider (and HMDB, for endogenous substrates). Of these, only 28 were endogenous substrates; the rest comprised a complex mixture of xenobiotic compounds, including pharmaceuticals, personal care products, industrial chemicals, pesticides, and steroids. Importantly,