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

Abstract

Non-targeted analysis (NTA) methods are increasingly used to discover chemicals 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, 1,269 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). An unblinded evaluation was then performed 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, *

In silico MS/MS spectra for identifying unknowns: a critical examination using CFM-ID algorithms and ENTACT mixture samples

“…Identification confidence levels [2] for each match of experimental data to a corresponding database/library entry are shown alongside the specified match in the workflow (right). Our previously reported results for the ENTACT mixtures were based on matching feature data to mass lists, formula lists, and reference MS2 libraries (highlighted in blue) [23]. The current examination incorporates searching against CFM-ID predicted spectra (highlighted in purple).…”

“…Sample preparation and analysis procedures have been previously described [23]. was used along with mobile phases consisting of 0.4 mM ammonium formate buffer in water and methanol.…”

“…In a previous analysis of the ENTACT mixtures, initial substance identification was performed without the use of individual reference standards. Thus, for any given spiked substance, determination of presence vs. absence could not be made with absolute certainty (i.e., Schymanski et al level 1) [23]. Features that could be linked to spiked substances with enough diagnostic evidence (e.g., MS1 and MS2 data corroborating an identification at the "probable structure" level [2]) were classified as "passes," indicating that there was strong evidence of their presence.…”

“…Within EPA's Office of Research and Development (ORD), initial analysis of the ENTACT mixtures has been performed and results of self-evaluation reported [23]. The purpose of the current article is to describe the incorporation of CFM-ID predicted spectra into the existing EPA workflow, and to evaluate overall method performance using the ENTACT mixture data.…”

High-resolution mass spectrometry to complement monitoring and track emerging chemicals and pollution trends in European water resources

“…Powerful LC-HRMS-and GC-HRMS-based screening methods are increasingly available [8][9][10][11][12]. Thus, this paper wants to encourage monitoring practitioners, water managers, and policy makers to consider these new techniques to achieve a more holistic water quality assessment and to enhance awareness on the multifold potential to make abatement and management of water pollution more efficient.…”