Improving the Screening Analysis of Pesticide Metabolites in Human Biomonitoring by Combining High-Throughput In Vitro Incubation and Automated LC–HRMS Data Processing

Abstract

There is a current need to monitor human exposure to a large number of pesticides and other chemicals of emerging concern (CECs). This requires screening analysis with high confidence for these compounds and their metabolites in complex matrices, which is hampered by the fact that no reference standards are available for most metabolites. We address this challenge by a high-throughput workflow based on incubation of pesticides (or other CECs) with human liver S9, followed by solid-phase extraction, liquid chromatography−high-resolution mass spectrometry (LC−HRMS) analysis, and automated data processing to generate a database (retention time, precursor m/z, and MS 2 spectral library) for the annotation in human samples. The metabolite prioritization consists of statistical comparisons and mass defect and m/z range filtering to obtain a subset of probable phase I metabolites, for which molecular formulas and likely metabolic transformation are retrieved. We tested the workflow on 22 pesticides, for which we could determine 91 metabolite molecular formulas which are only partly covered by the literature and/or predicted by in silico metabolization. Our workflow allows for an efficient generation of metabolite reference information, which can be used directly for annotating LC−HRMS data from human samples. A full structure elucidation of individual metabolites can be limited to those being actually present in human samples.

In silico deconjugation of glucuronide conjugates enhances tandem mass spectra library annotation of human samples

“…The organic phase was removed under a gentle nitrogen stream at room temperature. A clean-up procedure was applied including solid-phase extraction (Strata-X Microelution SPE, Phenomenex, Aschaffenburg, Germany) with a method described elsewhere [ 23 ].…”

Wastewater-based epidemiology for the assessment of population exposure to chemicals: The need for integration with human biomonitoring for global One Health actions

“…These are primarily: selection of specific human metabolic biomarkers, characteristics of urban water cycle including sewer systems (population size and mobility in a WWTP catchment, flow rate), and understanding the fate of biomarkers (stability, biotransformation, sorption). Novel screening approaches for human biomonitoring of a multitude of contaminant metabolites in urine based on high-throughput in vitro incubation, suspect and non-target Liquid Chromatography and High Resolution Mass Spectrometry (LC-HRMS) screening involving in silico deconjugation and automated data evaluation [23] , [24] create new opportunities to discover new relevant biomarkers providing insights to so far unknown exposures in both HBM as well as WBE.…”

Improving Wastewater-Based Epidemiology for New Psychoactive Substance Surveillance by Combining a High-Throughput In Vitro Metabolism Assay and LC−HRMS Metabolite Identification

“…In vitro metabolism bioassays have been proposed as a potential means to expand the capabilities of wastewater-based epidemiology (Lai et al, 2015;Lopardo et al, 2017;Reid et al, 2014). For example, external metabolization systems like the liver S9 fraction in vitro bioassay have been developed (Shao et al, 2020) and refined for high-throughput application (Escher et al, 2020;Huber et al, 2021;Villeneuve et al, 2019). Metabolism is also an important determinant of toxicity since chemicals can be activated or deactivated (detoxified) by, for example, Phase 1 cytochrome P450 monooxygenase (CYP) enzymes (Yu, 2020) and https://doi.org/10.26434/chemrxiv-2023-mvc6g ORCID: https://orcid.org/0000-0003-2724-9183 Content not peer-reviewed by ChemRxiv.…”

“…The combination of an S9 fraction in vitro metabolism bioassay with LC-HRMS was recently demonstrated as a high-throughput workflow for production of pesticide metabolites to generate a LC-HRMS database consisting of retention time, precursor ion and fragments (Huber et al, 2021). By combining the in vitro assay on parent NPS drugs with HRMS screening of the metabolism products, potential metabolites of NPS can be identified, and subsequently tested for use in WBE.…”