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Using prepared mixtures of ToxCast chemicals to evaluate non-targeted analysis (NTA) method performance


Non-targeted analysis (NTA) methods are increasingly used to discover contaminants of emerging concern (CECs), but the extent to which these methods can support exposure and health studies remains to be determined. EPA’s Non-Targeted Analysis Collaborative Trial (ENTACT) was launched in 2016 to address this need. As part of ENTACT, 1269 unique substances from EPA’s ToxCast library were combined to make ten synthetic mixtures, with each mixture containing between 95 and 365 substances. As a participant in the trial, we first performed blinded NTA on each mixture using liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS). We then performed an unblinded evaluation to identify limitations of our NTA method. Overall, at least 60% of spiked substances could be observed using selected methods. Discounting spiked isomers, true positive rates from the blinded and unblinded analyses reached a maximum of 46% and 65%, respectively. An overall reproducibility rate of 75% was observed for substances spiked into more than one mixture and observed at least once. Considerable discordance in substance identification was observed when comparing a subset of our results derived from two separate reversed-phase chromatography methods. We conclude that a single NTA method, even when optimized, can likely characterize only a subset of ToxCast substances (and, by extension, other CECs). Rigorous quality control and self-evaluation practices should be required of labs generating NTA data to support exposure and health studies. Accurate and transparent communication of performance results will best enable meaningful interpretations and defensible use of NTA data.

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The authors thank Annette Guiseppi-Elie, Jennifer Orme-Zavaleta, and Russell Thomas for supporting ENTACT; Katherine Coutros for her assistance in acquiring ToxCast substances, Kamel Mansouri for his role in developing and implementing MS-Ready processing algorithms; Risa Sayre for her assistance in comparing spiked substances against compounds in Agilent reference libraries; and Sarah Laughlin, Aurelie Marcotte, Dawn Mills, James McCord, Mark Strynar, and Carol Ball (Agilent Technologies) for their contributions to the methods used for sample analysis and data processing. The authors further thank James McCord and Mark Strynar for their thoughtful reviews of this manuscript.


The United States Environmental Protection Agency (U.S. EPA), through its Office of Research and Development (ORD), funded and managed the research described here. Partial support for this work was provided by an award from ORD’s Pathfinder Innovation Program. The work has been subjected to Agency administrative review and approved for publication. Randolph Singh and Andrew McEachran were supported by an appointment to the Internship/Research Participation Program at the Office of Research and Development, U.S. Environmental Protection Agency, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and EPA.

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Correspondence to Jon R. Sobus.

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Sobus, J.R., Grossman, J.N., Chao, A. et al. Using prepared mixtures of ToxCast chemicals to evaluate non-targeted analysis (NTA) method performance. Anal Bioanal Chem 411, 835–851 (2019).

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  • Non-targeted analysis
  • ToxCast
  • Exposome